Chitosan-derived compositions

ABSTRACT

The present invention relates generally to therapeutic compositions comprising chitosan-derived compositions used in connection with methods for treating neoplasms, such as for instance, malignant lung, thyroid and kidney neoplasms, and other types of malignant neoplasms, and other medical disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/028,221, filed Jul. 5, 2018, which is a Continuation-in-Part(C-I-P) application of previously filed U.S. patent application Ser. No.14/372,586, filed on Jul. 16, 2014, which is a 371 national phase entryfrom PCT application serial number PCT/US2013/021903, filed on Jan. 17,2013 and which herein claims priority to U.S. provisional patentapplication Ser. No. 61/588,783, entitled “Chitosan-Derived Biomaterialsand Applications Thereof” filed on Jan. 20, 2012, the entire contents ofwhich are incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates generally to therapeutic compositionscomprising chitosan-derived compositions used in connection with methodsfor treating neoplasms, such as, malignant lung, breast, prostate, skin,thyroid and kidney neoplasms, and other types of malignant neoplasms,and other medical disorders.

BACKGROUND OF THE INVENTION

Chitosan is a derivative of chitin, a compound usually isolated from theshells of some crustaceans such as crab, lobster and shrimp. Chitin is alinear homopolymer composed of N-acetylglucosamine units joined by β 1→4glycosidic bonds. Chitin, chitosan (partially deacetylated chitin) andtheir derivatives are endowed with interesting chemical and biologicalproperties that have led to a varied and expanding number of industrialand medical applications. Glycated chitosan, described in U.S. Pat. No.5,747,475 (“Chitosan-Derived Biomaterials”), which is hereinincorporated by reference, is one such chitosan derivative.

Cancer can develop in any tissue of any organ at any age. Once anunequivocal diagnosis of cancer is made, treatment decisions becomeparamount. Though no single treatment approach is applicable to allcancers, successful therapies must be focused on both the primary tumorand its metastases. Historically, local and regional therapy, such assurgery or radiation, have been used in cancer treatment, along withsystemic therapy, e.g., chemotherapy drugs. Despite some success,conventional treatments are not effective to the degree desired, and thesearch has continued for more efficacious therapies. There is clearly asignificant unmet need for more efficient cancer therapies.

Conventional glycated chitosan preparations, as described in U.S. Pat.No. 5,747,475 (“Chitosan-Derived Biomaterials”), have shown significantefficacy as an immunoadjuvant in the treatment of metastatic tumormodels in animals.

However, conventional glycated chitosan preparations, when dispersed,suspended or dissolved in aqueous solutions are often very difficult toinject or dispense in the biomedical applications to which they are put.Moreover, conventional glycated chitosan preparations, as described inU.S. Pat. No. 5,747,475 (“Chitosan-Derived Biomaterials”), are nearlyimpossible to sterile filter, rendering them unsuitable for industrialmanufacturing according to Current Good Manufacturing Practices (cGMP),and therefore unsuitable for human use. It is thus an object of thepresent invention to provide improved viscoelastic glycated chitosanpreparations which are far less subject to the above-noteddisadvantages.

SUMMARY OF THE INVENTION

According to one embodiment, the present invention relates generally totherapeutic formulations comprising chitosan-derived compositions usedin connection with methods for treating neoplasms and other medicaldisorders.

According to another embodiment, the present invention provides aviscoelastic glycated chitosan polymer of Formula 1:

wherein:

n is an integer of from about 5 to about 6900 for a molecular weightrange of 1000 to 1,500,000 Daltons; and the degree of glycation of freeamino groups of the chitosan polymer ranges between about one tenth ofone percent to about thirty percent.

According to another embodiment, the present invention provides aninjectable pharmaceutical composition comprising: a sterile filteredaqueous mixture of the viscoelastic glycated chitosan polymer of Formula1,

wherein: n is an integer of from about 5 to about 2300 for a molecularweight range of 1000 to less than 500,000 Daltons; and the degree ofglycation of free amino groups of the chitosan polymer ranges betweenabout one tenth of one percent to about thirty percent, in which thesterile filtered aqueous mixture has a pH from between 5 to about 7; andthe sterile filtered aqueous mixture having about one percent by weightof the viscoelastic glycated chitosan polymer dissolved therein so thatthe sterile filtered aqueous mixture has a viscosity from about onecentistokes to approximately one hundred centistokes measured at about25 degrees Celsius.

Additional aspects and/or advantages of the invention will be set forthin part in the description which follows and, in part, may be learned bypractice of the invention.

BRIEF DESCRIPTION OF THE FIGURES

These and/or other aspects and advantages of the invention will becomeapparent and more readily appreciated from the following description ofthe embodiments, taken in conjunction with the accompanying drawings ofwhich:

FIG. 1 depicts one conventional example of glycated chitosan, e.g.,galactochitosan.

FIG. 2 depicts one exemplary structure of viscoelastic glycated chitosanof the present invention, where the deacetylation of the parent chitosanis 80%, and the glycation of total available deacetylated amino groupsis 12.5%.

FIG. 3 depicts a graph that shows viscosity (in Cp: y-axis) vs.molecular weight (in kDa; x-axis) in samples of GC with molecularweights ranging from 100 kDa to 1,500 kDa).

FIG. 4 depicts a graph that shows the percent of GC in solution of thesamples used in the viscosity experiment.

FIG. 5 depicts a graph that shows the survival rates followinginterstitial laser-assisted immunotherapy with 0.2 ml of GC.

FIG. 6 depicts a graph that shows the effect of tumour-localizedglycated chitosan treatment on the response to mTHPC-based PDT in mouseLine 1 tumors. In this graph: GC=Glycated chitosan;mTHPC=Meso-substituted tetra (meta-hydroxy-phenyl) chlorin; andPDT=Photodynamic therapy.

FIGS. 7A-7C depict graphs that show rat survival rates followingtreatment with one, two, or three components of the laser-assistedimmunotherapy system. In these graphs, GC=1.0% glycated chitosan; andICG=0.25% indocyanine green.

FIG. 8 depicts a graph that shows rat survival curves in the adoptiveimmunity transfer experiments using rat splenocytes as immune cells. Inthis graph, Group A=Results from tumor control rats; group B=Resultsfrom rats injected with tumor cells admixed with spleen cells from anuntreated tumor-bearing rat; Group C=Results from rats injected withtumor cells admixed with spleen cells from laser-assisted immunotherapysuccessfully treated rat; Group D=Results using spleen cells from anaïve rate. Note: Data collected from 2 separate experiments werecombined and plotted together.

FIG. 9 is a Table showing recirculation data from Study #VAL-AM-000754-Bfor IP-001 Drug Product (an embodiment of GC).

FIG. 10 is a graph showing comparative filtration rate data for various1% solutions of GC.

FIG. 11 illustrates particle size data for three GC solutions.

Still other objects and advantages of preferred embodiments of thepresent invention will become readily apparent to those skilled in thisart from the following detailed description, wherein there is describedcertain preferred embodiments of the invention, and examples forillustrative purposes.

DETAILED DESCRIPTION

The invention relates generally to therapeutic formulations comprisingchitosan-derived compositions used in connection with methods fortreating neoplasms and other medical disorders. It is to be understoodthat all references cited herein are incorporated by reference in theirentirety.

Reference will now be made in detail to certain embodiments of thepresent invention, examples of which are illustrated in the accompanyingdrawings, wherein like reference numerals refer to the like elementsthroughout. It is to be understood that the invention is capable ofmodifications in various obvious respects, all without departing fromthe spirit and scope of the invention. Accordingly, the descriptionshould be regarded as illustrative in nature, and not as restrictive.

Glycated Chitosan

Glycated chitosan is a product of the glycation (i.e., non-enzymaticglycosylation) of free amino groups of chitosan, followed bystabilization by reduction. Glycation endows the chitosan withadvantageous solubility and viscosity characteristics which facilitatethe use of the derivative in conjunction with laser-assistedimmunotherapy and other applications of the derivative. The glycation ofchitosan also renders the chitosan more hydrophilic whereby more wateris absorbed and retained by the polymer than would otherwise be thecase.

In accordance with preferred embodiments of the present invention, achitosan-derived biomaterial comprises a linear homopolymer ofdeacetylated chitin (chitosan), wherein the deacetylated chitin has anumber of otherwise free amino groups bonded to a carbonyl group of areducing monosaccharide or oligosaccharide to form glycated chitosan.Glycated chitosan can thus be obtained as the reaction product betweenthe carbonyl group of a reducing monosaccharide or oligosaccharide andthe free amino groups of deacetylated chitin. Thus, the term “glycatedchitosan” as used herein is intended to refer to a product of theglycation, i.e., non-enzymatic glycosylation, of free amino groups ofchitosan, followed by stabilization by reduction. Generally speaking,glycation (or non-enzymatic glycosylation) is intended to refer to aprocess that occurs when a sugar molecule, such as fructose or glucose,binds to a substrate, such as a protein or lipid molecule, without thecontributing action of an enzyme. One such example is the non-enzymaticreaction of a sugar and an amine group of a protein to form aglycoprotein.

Glycated chitosan, thus generally includes the products resulting fromthe reaction between the free amino groups of chitosan and the carbonylgroups of reducing monosaccharides and/or oligosaccharides. The productsof this reaction, which mainly are a mixture of Schiff bases (i.e. thecarbon atom from the carbonyl group is now doubly bonded to the nitrogenfrom the free amine releasing one molecule of water) and Amador products(i.e. the carbon atom of said carbonyl group is singly bonded to thenitrogen atom of said amino group while an adjacent carbon atom isdouble bonded to an oxygen atom) may be used as such or afterstabilization by reduction with hydrides, such as boron-hydride reducingagents, for example NaBH₄, NaBH₃CN, NaBH(OAc)₃, etc, or by exposure tohydrogen in the presence of suitable catalysts.

The presence of primary and secondary alcohol groups, and of primaryamino groups in chitosan, facilitate a number of approaches for chemicalmodifications designed mainly to achieve their solubilization and toimpart special properties for specific applications.

Solubilization of chitin and chitosan can be achieved by partialhydrolysis to oligosaccharides. For chitosan, treatment with a varietyof acids, both organic and inorganic, leads to the formation of watersoluble chitosonium salts by protonation of the free amino groups.Additional modifications of the amino groups include the introduction ofchemical groups such as carboxymethyl, glyceryl, N-hydroxybutyl andothers. Glycation, i.e., non-enzymatic glycosylation of the free aminogroups of chitosan, followed by stabilization by reduction, offers apreferred approach for the preparation of various pharmaceuticalformulations utilized in the present invention.

For illustrative purposes, one conventional example of glycatedchitosan, e.g., galactochitosan, is shown in FIG. 1 , which is alsodescribed and illustrated in U.S. Pat. No. 5,747,475.

FIG. 1 is an exemplary structure of conventional glycated chitosan,where the molecular weight is approximately 1,500,000 Daltons and allamino groups are glycated.

U.S. Pat. No. 5,747,475 is very limited in its description and describesonly one specific galactochitosan in terms of molecular weight;specifically, U.S. Pat. No. 5,747,475 only describes galactochitosanwith a molecular weight of 1500 kDa.

Unlike the conventional 1500 kDa galactochitosan described in U.S. Pat.No. 5,747,475, it is to be clearly understood that the glycated chitosanof the present invention as described herein is intended to includeglycated chitosan having a molecular weight less than 1500 kDa.Moreover, unlike conventional chitosans, the glycated chitosans of thepresent invention is a completely different and novel composition ofmatter with a number of surprisingly unexpected properties, benefits andadvantages, including unexpectedly beneficial viscoelastic properties.

The glycated chitosan of the present invention is in the form of aSchiff base, an Amadori product, or preferably, in their reducedsecondary amine or alcohol, respectively. In another embodiment, theglycated chitosan includes a carbonyl reactive group. It is preferredthat glycated chitosan of the present invention is obtained by reactingchitosan with a monosaccharide and/or oligosaccharide, preferably in thepresence of an acidifying agent, for a time sufficient to accomplishSchiff base formation between the carbonyl group of the sugar and theprimary amino groups of chitosan (also referred to herein as glycationof the amino group) to a degree whereby about 0.1% to about 30% (andmost preferably above 2%) glycation of the amino groups of the chitosanpolymer is achieved. This is preferably followed by stabilization byreduction of Schiff bases and of their rearranged derivatives (Amadoriproducts) to the secondary amines or alcohols.

The present invention is the first demonstration whereby about 0.1% toabout 30% (and most preferably above 2%) glycation of the chitosanpolymer is achieved. Contrary to the present invention, others havefailed to achieve or recognize this significant result. Thus, accordingto a preferred embodiment, the present invention provides a viscoelasticglycated chitosan formulation, consisting essentially of glycatedchitosan polymer, wherein the glycated chitosan polymer has a molecularweight between about 50,000 Daltons to about 1,500,000 Daltons, andfurther wherein the glycated chitosan polymer possesses from about onetenth of a percent to about thirty percent glycation of its otherwisefree amino groups.

The products resulting from the non-enzymatic glycosylation of freeamino groups of chitosan are thus mainly a mixture of Schiff bases, i.e.the carbon atom of the initial carbonyl group double bonded to thenitrogen atom of the amino group (also known as the imine functionalgroup), and Amadori products, i.e. the carbon atom of the initialcarbonyl group bonded to the nitrogen atom of said amino group by asingle bond while an adjacent carbon atom is double bonded to an oxygenatom forming a ketone group. These products (resulting from thenon-enzymatic glycosylation process) may be used as such, or afterstabilization by reduction with hydrides, such as boron-hydride reducingagents, for example NaBH₄, NaBH₃CN, NaBH(OAc)₃, etc, or by exposure tohydrogen in the presence of suitable catalysts.

Chitosan deamination with nitrous acid can be used to generate reducingaldoses and oligosaccharides suitable for the glycation of chitosan.Deamination of the deacetylated glucosaminyl residues by nitrous acidresults in the selective cleavage of their glycosidic bonds with theformation of 2,5-anhydro-D-mannose residues. Depending on thecomposition of specific areas of the chitosan chain, the anhydro hexosecould be released as the monosaccharide, or occupy the reducing end ofan oligosaccharide. Release of free N-acetylglucosamine could also occurfrom some regions of the chitosan chain. Similar treatment ofN-deacetylated glycoproteins and glycolipids can be utilized to obtainoligosaccharides of defined chemical composition and biological activityfor special preparations of glycated chitosan.

Various products obtained by chitosan glycation will be utilized as suchor reacted with other natural or synthetic materials, e.g., reaction ofaldehyde-containing derivatives of glycated chitosan with substancescontaining two or more free amino groups, such as on the side chains ofamino acids rich in lysine residues as in collagen, on hexosamineresidues as in chitosan and deacetylated glycoconjugates, or on naturaland synthetic diamines and polyamines. This is expected to generatecrosslinking through Schiff base formation and subsequentrearrangements, condensation, dehydration, etc. Stabilization ofmodified glycated chitosan materials can be made by chemical reductionor by curing involving rearrangements, condensation or dehydration,either spontaneous or by incubation under various conditions oftemperature, humidity and pressure. The chemistry of Amadorirearrangements, Schiff bases and the Leukart-Wallach reaction isdetailed in The Merck Index, Ninth Edition (1976) pp. ONR-3, ONR-55 andONR-80, Library of Congress Card No. 76-27231, the same beingincorporated herein by reference. The chemistry of nucleophilic additionreactions as applicable to the present invention is detailed in Chapter19 of Morrison and Boyd, Organic Chemistry, Second Edition (eighthprinting 1970), Library of Congress Card No. 66-26695, the same beingincorporated herein by reference.

As further described herein, particular types (e.g., particular types ofreducing sugars) and degrees of glycation have surprisingly been foundto endow the chitosan with unexpected and advantageous solubilitycharacteristics which facilitate the use of the glycated chitosan inconjunction with laser-assisted immunotherapy and other therapeuticapplications. The glycation of chitosan also advantageously renders thechitosan more hydrophilic whereby more water is absorbed and retained bythe polymer than would otherwise be the case. The D-galactose derivativeof chitosan is particularly preferred insofar as D-galactose has arelatively higher naturally occurring incidence of its open chain form.The glycated chitosan may be prepared in any number of suitableformulations including, for example, a powder form, as a viscousformulation, or in any other suitable form.

In accordance with other preferred embodiments of the invention,chitosan may be non-enzymatically glycated utilizing any of a number ofthe same or different reducing sugars, e.g., the same or differentmonosaccharides and/or oligosaccharides. Examples of such monosaccharideglycosylation agents are the more naturally occurring D-trioses,D-tetroses, D-pentoses, D-hexoses, D-heptoses, and the like, such asD-glucose, D-galactose, D-fructose, D-mannose, D-allose, D-altrose,D-Idose, D-talose, D-fucose, D-arabinose, D-gulose, D-hammelose,D-lyxose, D-ribose, D-rhamnose, D-threose, D-xylose, D-psicose,D-sorbose, D-tagatose, D-glyceraldehyde, dihydroxyacetone, D-erythrose,D-threose, D-erythrulose, D-mannoheptulose, D-sedoheptulose and thelike. Suitable oligosaccharides include the fructo-oligosaccharides(FOS), the galacto-oligosaccharides (GOS), the mannan-oligosaccharides(MOS) and the like.

Preferred Viscoelastic Properties

Conventionally produced chitosan products, when dispersed, suspended ordissolved in aqueous solutions are very difficult to produce accordingto GMP standards, and have a number of disadvantages in terms ofadministration and other uses.

Preferred embodiments of the present invention overcome the long unmetneeds for improved therapeutic chitosan products by providing improvedviscoelastic glycated chitosan preparations which are not subject to thedisadvantages of conventional approaches.

The term “viscoelastic” as used herein refers to the viscosity of aparticular composition, preparation or formulation. Viscosity is wellunderstood as a measure of the resistance of a fluid which is beingdeformed by either shear stress or tensile stress. In other words,viscosity describes a fluid's internal resistance to flow and may bethought of as a measure of fluid friction.

a. Unexpected Improvements in Injectability of GC Preparations

It has been surprisingly and unexpectedly discovered that theinjectability of formulations of glycated chitosan (GC), for instancesolutions or suspensions, is nonobviously dependent upon the viscosityand rheological properties of the GC. These properties are, in turn,highly dependent upon the molecular weight of the GC, the degree ofpolymerization of the chitin parent to the chitosan, the degree ofdeacetylation of the chitin parent, and the degree of glycation of thechitosan. These latter properties determine the degree of entanglementof the polymer chains of the GC as well as the degree of intramolecularhydrogen bonding occasioned by the number and nature of the substituentspresent on the GC molecule (i.e., acetyl and saccharide), both of whichcontribute significantly to the viscosity and other rheologicalproperties of solutions prepared therefrom.

It has been surprisingly and unexpectedly discovered that the improvedviscoelastic glycated chitosan preparations of the present inventionpossess numerous advantages, for instance, (i) administration of anon-toxic preparation for treatment of neoplasm in a patient; (ii) farsuperior injectability (e.g., through different gauge needles) in aclinical setting as compared to conventional treatments; (iii) improvedsterile filtration of the viscoelastic preparations; and (iv) a lesspainful and thus an improved treatment option for patients. The term“injectability” as used herein refers to the ease with which aformulation or preparation, for instance, a formulation comprisingglycated chitosan (GC), is injected into a subject.

According to one preferred embodiment, the invention provides aninjectable viscoelastic preparation comprising approximately 1 percentby weight of the above-described glycated chitosan dispersed, suspendedor dissolved in an aqueous solution.

Preferred embodiments of the invention include preparations of glycatedchitosan, including for instance solutions or suspensions, that have aviscosity that renders the preparations readily injectable via a needlewith a relatively large needle gauge (G), thus reducing pain anddiscomfort for the subject. Preferred examples of relatively large gaugeneedles include needles that have the following dimensions: a nominalinner diameter of from about 0.337 mm (23 G) to about 0.210 mm (27 G).

According to one example, a viscoelastic glycated chitosan preparationis administered via injection using an injection needle having adiameter of about 20 G to about 22 G, and an effective length of a tubeof the injection needle is about 1,000 mm or more such that the inflowrate of the injectable preparation, when injected at a pressure of abouttwo to about three atmospheres through said injection needle, rangesfrom about 0.05 ml/second to 0.1 ml/second. According to anotherexample, a viscoelastic glycated chitosan preparation can also beadministered via injection using an injection needle having a diameterof about 25 G to about 27 G. It is also to be understood that aviscoelastic glycated chitosan preparation according to the presentinvention can also be administered using any other suitable gauge needleor instrument.

It has been surprisingly found that the viscoelastic glycated chitosanpreparations of the present invention, for instance, solutions orsuspensions, are injectable at a relatively wide range of concentrationsthrough catheters or needles of the most commonly used gauges.

It has also been discovered that these improved viscoelastic glycatedchitosan preparations (i.e., by improving the viscosity and rheologicalproperties of the glycated chitosan compositions) also unexpectedlyimprove the overall ease of administration of the preparation to asubject; the efficiency of administration by the individualadministering the formulation (for example, nurse, physician, or otherhealthcare practitioner), and the compliance and efficacy of theglycated chitosan formulations may also be enhanced.

b. Unexpected Improvements in Manufacturing and Filtration

It has also been surprisingly found that sterile filtration isunexpectedly improved using the improved viscoelastic glycated chitosanpreparations of the present invention. Conventional glycated chitosanpreparations, as described in U.S. Pat. No. 5,747,475 (“Chitosan-DerivedBiomaterials”), was shown to be very difficult to sterile filter througha 0.22 um sterile filter, which renders it unsuitable for commercialcGMP manufacturing. In contrast, the improved viscoelastic glycatedchitosan, which was discovered to have nonobvious rheologicalproperties, was shown to be highly suitable for sterile filtration, cGMPmanufacturing, and human use.

Furthermore, it has been surprisingly found that diafiltration andultrafiltration is unexpectedly improved using the improved viscoelasticglycated chitosan preparations of the present invention. Conventionalglycated chitosan preparations were difficult to diafilter andultrafilter, causing the filter to clog, thus rendering it unsuitablefor commercial cGMP manufacturing. The improved viscoelastic glycatedchitosan, on the other hand, was highly suitable for diafiltration andultrafiltration, thus significantly improving the manufacturing process.

Exemplary Methods for Determination of Viscosity

Any number of suitable techniques in the chemical arts can be used toreliably and accurately determine viscosity of a glycated chitosanformulation.

It is to be understood that viscosity can be reliably measured withvarious types of instruments, e.g., viscometers and rheometers. Arheometer is used for those fluids which cannot be defined by a singlevalue of viscosity and therefore require more parameters to be set andmeasured than is the case for a viscometer. Close temperature control ofthe fluid is essential to accurate measurements, particularly inmaterials like lubricants, whose viscosity can double with a change ofonly 5° C.

In accordance with the present invention, the viscosity of a glycatedchitosan preparation can be determined according to any suitable methodknown in the art.

For instance, viscosity can be reliably measured in units of centipoise.The poise is a unit of dynamic viscosity in the centimeter gram secondsystem of units. A centipoise is one one-hundredth of a poise, and onemillipascal-second (mPa·s) in SI units (1 cP=10⁻² P=10⁻³ Pa·s).Centipoise is properly abbreviated cP, but the alternative abbreviationscps, cp, and cPs are also commonly seen. A viscometer can be used tomeasure centipoise. When determining centipoise, it is typical that allother fluids are calibrated to the viscosity of water.

Exemplary Determination of Viscosity of Glycated Chitosan Preparations

There are numerous factors that affect the viscosity of solutions and,in particular, solutions of polymers, other than molecular weight. Inthe case of glycated chitosan (GC) the injectability of solutions of GCis highly dependent upon the viscosity and rheological properties of theGC in solution. These properties are, in turn, highly dependent upon themolecular weight of the GC, the degree of polymerization of the chitinparent to the chitosan, the degree of deacetylation of the chitinparent, and the degree of glycation of the chitosan. These latterproperties determine the degree of entanglement of the polymer chains ofthe GC as well as the degree of intramolecular hydrogen bondingoccasioned by the number and nature of the substituents present on theGC molecule (i.e., acetyl and saccharide), both of which contributesignificantly to the viscosity of solutions prepared therefrom.

It has been surprisingly discovered that the improved viscosity andrheological properties of glycated chitosan preparations are, in turn,highly dependent upon particular physiochemical properties of theglycated chitosan. The term “physiochemical property” as used herein isintended to include, but is not limited to, any physical, chemical orphysical-chemical property of a molecular structure, such as glycatedchitosan. As described further herein, a few examples of thesephysiochemical properties are:

(i) the molecular weight of the glycated chitosan;

(ii) the degree of polymerization of the chitin parent to the chitosan;

(iii) the degree of deacetylation of the chitin parent; and

(iv) the degree of glycation of the chitosan.

Structural Formula 1

In one embodiment, there is provided a viscoelastic glycated chitosan ofFormula 1, as illustrated in FIG. 2 :

Formula 1 includes ten monomeric units, one of which being galactated,representing, in one example, 12.5% galactation. In one example, where5% galactation is present, the integer “n” ranges from about 5 to about6900, which represents a molecular weight range of 1000 to 1,500,00daltons. For a 250 kDa chain, there would be 1150 monomers. Given thatthe formula represents a semisynthetic biopolymer, the person ofordinary skill in the art will readily understand that the molecularweights are given as an average because in any formulation, there willbe polymer chains of larger and smaller chain length. In one specificexample, there is provided a viscoelastic glycated chitosan where themolecular weight is approximately 250 kDa, where the deacetylation ofthe parent chitosan is about 80%, and the glycation of total availabledeacetylated amino groups is about 12.5%. Specifically, in a preferredembodiment the molecular weight of the viscoelastic glycated chitosan isless than 500,000 Daltons.

(i) Molecular Weight of the Glycated Chitosan

Any number of suitable techniques in the chemical arts can be used toreliably and accurately determine the molecular weight (MW) of theglycated chitosan.

It is preferred that a viscoelastic glycated chitosan preparation isprepared as an injectable formulation comprising glycated chitosan witha molecular weight (MW) less than about 1500 kDa. Examples of preferredviscoelastic glycated chitosan preparations comprise glycated chitosanwith a molecular weight (MW) of between about 50 Kilodaltons (kDa) andabout 1500 kDa. Specifically, in a preferred embodiment the molecularweight is less than 500,000 Daltons.

In certain embodiments, a viscoelastic glycated chitosan preparationcomprises glycated chitosan with a molecular weight (MW) of betweenabout 100 kDa and about 1000 kDa; and more preferably, between about 100kDa and about 300 kDa. Specifically, in a preferred embodiment themolecular weight is less than 500,000 Daltons.

In certain specific embodiments, n is an integer of from about 5 toabout 6900 for a molecular weight range of 1000 to 1,500,000 Daltons. Inone example, the viscoelastic glycated chitosan polymer has a molecularweight between about 50,000 Daltons to about 1,500,000 Daltons. Inanother example, the viscoelastic glycated chitosan polymer has amolecular weight between about 190,000 Daltons to about 310,000 Daltons.In yet another example, the viscoelastic glycated chitosan polymer, themolecular weight is approximately 250,000 Daltons. Specifically, in apreferred embodiment the molecular weight is less than 500,000 Daltons.

Various techniques can be used to accurately determine molecular weight.

The invention encompasses chitosan-derived compositions comprisingderivatives of chitosan which are water-soluble or water-dispersible. Inaccordance with the present invention, it has also been surprisinglyfound that in certain embodiments, with increasing molecular weight(MW), more water is required to solubilize the glycated chitosan (GC).This in turn means less amount of the water is “free”, i.e. nothydrogen-bonded to the GC (assuming no additional water is added to thesolution), which in itself contributes to higher viscosity. As shown inexample 3 below, this result has been unexpectedly found to add to theviscosity increase that is given by the increasing size of the molecule,giving an exponential (or something similar), rather than a linearrelationship between viscosity and MW (when concentration is compensatedfor).

(ii) Degree of Polymerization (DP) of the Chitin Parent to the Chitosan

The degree of polymerization (DP) of the chitin parent to the chitosancan be reliably and accurately determined according to any number ofsuitable methods or techniques known in the chemical arts.

In one approach, it is preferred that the degree of polymerization (DP)is determined by dividing the molecular weight of the chitosan by themolecular weight of the glucosamine link.

(iii) Degree of Deacetylation of the Chitin Parent

Another physiochemical property is the degree of deacetylation of thechitin parent. Any number of suitable techniques in the chemical artscan be used to reliably and accurately determine the degree ofdeacetylation of the chitin parent.

NMR is one technique that can be used to determine the degree ofdeacetylation of chitin or chitosan.

(iv) Degree of Glycation of the Chitosan

Any number of suitable techniques in the chemical arts can be used toreliably and accurately determine the degree of glycation of thechitosan.

NMR is one technique that can be used to detect and measure the bondingof monosaccharides and/or oligosaccharides to the chitosan polymer.

C/N elemental combustion analysis is another technique that can be usedto determine the percent glycation of the glycated chitosan by means ofcomparing the C/N ratio of glycated chitosan vs. the parent chitosan.

Enzymatic digestion coupled with HPLC is yet another technique that canbe used to determine percent glycation.

It is to be understood that other suitable analytical methods andinstrumentation can also be used for simultaneous detection, measurementand identification of multiple components in a sample, e.g., forsimultaneous detection, measurement and identification of glycated andnon-glycated chitosan in a sample.

Colorimetric measurement of chemicals bound to remaining free aminogroups, such as via a ninhydrin reaction, can be used to assess thedegree of glycation.

It has thus been found that glycated chitosans having preferredmolecular weights, degrees of polymerization of the chitin parent to thechitosan, degrees of deacetylation of the chitin parent, and degrees ofglycation of the chitosan enable improved preparation of glycatedchitosan solutions which are injectable at a relatively wide range ofconcentrations of the glycated chitosan through catheters or needles ofcommonly used gauges.

Preferred Methods of Preparing Glycated Chitosan

Still other embodiments of the invention relate to methods for thepreparation of glycated chitosan formulations. Glycated chitosan ispreferably obtained by reacting chitosan with a monosaccharide and/oroligosaccharide, preferably in the presence of an acidifying agent, fora time sufficient to accomplish Schiff base formation between thecarbonyl group of the sugar and the primary amino groups of chitosan(also referred to herein as glycation of the amino group) to a degreewhereby at least some percentage (for example, two percent or higher)glycation of the chitosan polymer is achieved. This is preferablyfollowed by stabilization by reduction of Schiff bases and of theirrearranged derivatives (Amadori products) to their secondary amines oralcohols, respectively. NMR tracings can be used to verify the bondingof the monosaccharides and/or oligosaccharides to the chitosan polymer,whereas chemical measurement of remaining free amino groups, such as viaa ninhydrin reaction, can be used to assess the degree of glycation.

In preferred embodiments, conditions can be adjusted as needed toimprove desired results during the manufacture of glycated chitosan. Forinstance, it has been unexpectedly discovered, in accordance with thepresent invention, that improvements in the manufacture of glycatedchitosan can be achieved by controlling the pH conditions, as describedfor instance in Example 4.

According to one example of preparation of glycated chitosan for use inthe present invention, approximately three grams of a reducingmonosaccharide (e.g., glucose, galactose, ribose), or an equivalentamount of a reducing oligosaccharide, is dissolved in 100 ml ofdistilled water under gentle magnetic stirring in an Erlenmeyer flask.Then approximately one gram of chitosan is added, and thereaftersuitable process steps can then be performed to yield the glycatedchitosan preparation with desired viscoelastic properties and withdesired purity characteristics.

One exemplary method for industrial-scale production of chitosanInvolves the following four steps: demineralization (DM),deproteinization (DP), decoloration (DC) and deacetylation (DA). Chitinextraction from, e.g., crustacean shells is carried out by analkali-acid treatment. Samples are deproteinized by treating withalkaline formulation, demineralized with acid and decolorized withorganic solvent (e.g., acetone), followed by bleaching (with, e.g.,sodium hypochlorite). Chitin deacetylation is carried out using, e.g.,sodium hydroxide formulation. The degree of polymerization of chitosanis adjusted by depolymerization; the most convenient procedures being(1) nitrous acid degradation in deuterated water. The reaction isselective, stoichiometric with respect to GlcN, rapid, and easilycontrolled, (2) depolymerization by acid hydrolysis, or (3) enzymaticdegradation with a commercial preparation (Pectinex Ultra Spl). Theenzymatic method yields shorter fragments with a higher proportion offully deacetylated chitooligomers. Conversely, acid hydrolysis of thestarting chitosan results in fragments with degrees of polymerization upto sixteen and more monoacetylated residues than with the enzymaticprocedure.

Polymerized Glycated Amino-Sugars

As described herein, chitosan (partially deacetylated chitin) is aderivative of chitin (a linear homopolymer composed ofN-acetylglucosamine units joined by β 1→4 glycosidic bonds).Chitosan-derived compositions thus comprise a homopolymer of partiallydeacetylated chitin, wherein the partially deacetylated chitin has anumber of otherwise free amino groups bonded to a carbonyl group of areducing monosaccharide or oligosaccharide creating an imine bond(Schiff Base) or related product (Amadori Rearrangement) and releasingone molecule of water.

Since chitin and chitosan are polymers of glucosamine, the presentinvention also contemplates non-enzymatically glycated glucosamine,e.g., glycated glucosamine monomers, or glycated glucosamine units. Inother words, the present invention also contemplates non-enzymaticglycation of amino-sugar monomers in general.

For instance, one example is a glycated glucosamine wherein theN-substituent is a galactose. It is preferred that glycation ofglucosamine monomers is performed after at least a percentage of theglucosamine monomers are initially deacetylated.

Moreover, the present invention also contemplates (1) polymers ofglycated glucosamine units (polymerized glycated amino-sugars), (2)combination polymers of glycated and non-glycated glucosamines, and (3)combinations of glycated and non-glycated glucosamine polymers wherein:

i) the percentage of non-deacetylated glucosamine monomers is from about1% to about 30%;

ii) the degree of polymerization (of the various combinations ofdeacetylated, non-deacetylated, glycated and non-glycated glucosamineunits) is from about x_(n)=300 to about x_(n)=8000, most preferablyabout x_(n)=1500; and/or

iii) the percent glycation of the free amino groups of the deacetylatedpolymerized glucosamine is from about 0.1% to about 30%.

The present invention also contemplates uses of polymerized glycatedglucosamine polymers that are the same or similar to uses of glycatedchitosan. These include, for instance, immunoadjuvant properties anduses, e.g., in the context of in situ cancer vaccines (inCVAX) such aslaser-assisted immunotherapy (LIT).

Exemplary Formulations and Applications

Examples of various types of pharmaceutically acceptable formulations orpreparations that can be used in accordance with the present inventioninclude, for instance, solutions, suspensions, and other types of liquidor semi-liquid formulations for injectability of the viscoelasticglycated chitosan preparations. For instance, the pharmaceuticallyacceptable formulations or preparations may include glycated chitosandispersed, suspended or dissolved in substantially aqueous formulations.By use of the term “substantially aqueous” it is to be understood thatthe formulations or preparations, in certain embodiments, may includesome percentage of one or more non-aqueous components, and one or morepharmaceutically acceptable excipients.

According to one example, a viscoelastic preparation is preferablyformulated as an aqueous solution possessing a pH from between about 5.0to about 7.

A viscoelastic preparation can also be formulated as an aqueous solutioncomprising a buffered physiological saline solution consistingessentially of glycated chitosan.

A viscoelastic preparation can also be formulated consisting essentiallyof glycated chitosan polymer, wherein the glycated chitosan polymerpossesses from about one tenth (0.1) of a percent to about thirty (30)percent glycation of its otherwise free amino groups.

According to a specific example, the glycated amino groups are presentfrom about one tenth of one percent to less than thirty percent ofavailable amino groups. According to another example, the viscoelasticglycated chitosan polymer includes glycated amino groups present from 1%to 8% of available amino groups. In yet another example, theviscoelastic glycated chitosan polymer includes glycated amino groupspresent from 3 to 6% of available amino groups. In still anotherexample, the viscoelastic glycated chitosan polymer includes glycatedamino groups present from about 0.5% to about 9.5% of available aminogroups,

In another embodiment, a viscoelastic preparation can be formulatedconsisting essentially of glycated chitosan (GC) polymer, wherein theglycated chitosan polymer possesses about two (2) percent glycation ofits otherwise free amino groups.

In another embodiment, a viscoelastic preparation can be formulatedconsisting essentially of glycated chitosan polymer, wherein theglycated chitosan polymer has a molecular weight between about 50,000 toabout 1,500,000 Daltons. Specifically, in a preferred embodiment themolecular weight is less than 500,000 Daltons.

Another example includes a viscoelastic GC preparation comprising aboutone (1) percent by weight of a glycated chitosan polymer dispersed in anaqueous solution, said aqueous solution having a viscosity of betweenabout one (1) to about one hundred (100) centistokes measured at about25 degrees Celsius.

Yet another example includes an aqueous solution having about onepercent by weight of glycated chitosan and from about one tenth (0.1) ofa percent to about thirty (30) percent glycation of otherwise free aminogroups of said glycated chitosan, wherein the aqueous solution has aviscosity from about one (1) centistokes to approximately one hundred(100) centistokes.

In yet another embodiment, a viscoelastic preparation can be formulatedconsisting essentially of glycated chitosan polymer, comprising about orabove one percent by weight of the glycated chitosan polymer dispersedin an aqueous solution, wherein the glycated chitosan polymer possessesabout two (2) percent glycation of its otherwise free amino groups, andwherein the aqueous solution has a viscosity suitable for ease ofinjectability and administration to a subject.

In yet another embodiment, a viscoelastic preparation can be formulatedconsisting essentially of glycated chitosan polymer, additionallycontaining one or more different viscoelastic materials miscible in anaqueous solution. Examples of suitable viscoelastic materials include,but are not limited to, hyaluronic acid, chondroitin sulfate andcarboxymethylcellulose.

The viscoelastic preparation can include glycated chitosan polymercomprising a monosaccharide bonded to an otherwise free amino group. Theglycated chitosan polymer can take any suitable form, such as a Schiffbase, an Amadori product or mixtures thereof. The glycated chitosanpolymer can also be in the form of a reduced Schiff base (secondaryamine), a reduced Amadori product (alcohol) or mixtures thereof.

The viscoelastic preparation can also be formulated wherein the glycatedchitosan polymer possesses a number of chemically modifiedmonosaccharide or oligosaccharide substituents. In one embodiment, themonosaccharide comprises galactose.

The inventive formulations or preparations preferably also containglycated chitosan in a physiologically compatible carrier.“Physiologically compatible” as used herein is to be understood to referto materials which, when in contact with tissues in the body, are notharmful thereto. The term is intended in this context to include, but isnot limited to, aqueous formulations (e.g., solutions) which areapproximately isotonic with the physiological environment of interest.Non-isotonic formulations (e.g., solutions) sometimes may also beclinically useful such as, for example dehydrating agents. Additionalcomponents of the inventive solutions may include various salts such as,for instance, NaCl, KCl, CaCl₂, MgCl₂ and Na based buffers.

The above and other objects are realized by the present invention,certain preferred embodiments of which relate to glycated chitosanpreparations having particular physiochemical properties that conferunexpected and surprisingly beneficial properties.

The present invention also encompasses a wide range of uses ofviscoelastic glycated chitosan preparations that have surprising andunexpected properties as immunoadjuvants, for instance, in connectionwith in situ autologous cancer vaccines, such as laser-assistedimmunotherapy for cancer, as described further herein.

Preferred embodiments of the invention provide immunoadjuvantscomprising an injectable viscoelastic preparation. It is thus anotherobject of the present invention to provide improved viscoelasticglycated chitosan preparations for other therapeutic applications,including therapeutic use as an immunoadjuvant and immunomodulator.

The present invention also encompasses various routes of administeringthe viscoelastic glycated chitosan immunoadjuvant formulations, such asvia injection. In a preferred approach, the immunoadjuvant is preferablyprepared as a formulation for injection into or around the tumor mass.It should be recognized however that other methods may be sufficient forlocalizing the immunoadjuvant in the tumor site. One such alternativedelivery means is conjugation of the immunoadjuvant to a tissue specificantibody or tissue specific antigen, such that delivery to the tumorsite is enhanced. Any one method, or a combination of varying methods,of localizing the immunoadjuvant in the tumor site is acceptable so longas the delivery mechanism insures sufficient concentration of theimmunoadjuvant in the neoplasm,

According to certain preferred embodiments, the present inventionprovides for various pharmaceutical formulations comprising viscoelasticglycated chitosan used in connection with in situ autologous cancervaccines (inCVAX), such as laser-assisted immunotherapy, photodynamiccancer therapy (PDT) and/or other tumor immunotherapy methods, asdescribed in further detail herein. It has been observed that it isdesirable to utilize glycated chitosan preparations having a suitableviscosity that enables their use as an injectable or other formulationas an immunoadjuvant in applications such as inCVAX and/or PDT and/ortumor immunotherapy methods. Such applications typically involveinjection of the viscoelastic glycated chitosan formulation into thecorpus of a patient. The term “immunoadjuvant” as used herein isintended to refer to any molecule, composition or substance that acts toenhance the immune system's response to an antigen; for instance,glycated chitosan which acts to enhance the immune system's response toa tumor antigen.

The immunoadjuvant composition can further include a tumor specificantibody conjugated to the glycated chitosan. The immunoadjuvantcomposition can also include a tumor specific antigen conjugated to theglycated chitosan. The glycated chitosan can further include a carbonylreactive group.

According to one preferred embodiment, the present invention provides animmunoadjuvant formulation that includes a suspension or a solution ofviscoelastic glycated chitosan. The viscoelastic glycated chitosan is inthis preferred embodiment used in connection with photothermal treatmentof a neoplasm without the use of a chromophore, where the light energyis delivered directly to the neoplasm. The light energy can be deliveredtopically if the neoplasm is accessible on the tissue surface (forexample melanoma), or is exposed by means of surgery. The light energycan also be delivered to the neoplasm by means of fiberoptics, forexample if the neoplasm is present below the tissue surface (for examplebreast cancer) and is not exposed through surgery.

According to another embodiment, and as described in further detailherein, the immunoadjuvant formulations of the present invention canfurther include a suitable chromophore. The selection of an appropriatechromophore is largely a matter of coordination with an acceptable laserwavelength of radiation. The wavelength of radiation used must, ofcourse, be complementary to the photoproperties (i.e., absorption peak)of the chromophore. Other chromophore selection criteria include abilityto create thermal energy, to evolve singlet oxygen and other activemolecules, or to be toxic in their own right such as cis-platinin. Inthe present invention, a preferred wavelength of radiation is805.+/−0.10 nm. The desired chromophores have strong absorption in thered and near-infrared spectral region for which tissue is relativelytransparent. Another advantage of this wavelength is that the potentialmutagenic effects encountered with UV-excited sensitizers are avoided.Nevertheless, wavelengths of between 150 and 2000 nm may prove effectivein individual cases. The preferred chromophore is indocyanine green.Other chromophores may be used, however, their selection being based ondesired photophysical and photochemical properties upon whichphotosensitization efficiency and photocytotoxicity are dependent.Examples of alternative chromophores include, but are not limited to,single walled carbon nanotubes (SWNT), buckminsterfullerenes (C₆₀),indocyanine green, methylene blue, DHE (polyhaematoporphrinester/ether), mm-THPP (tetra(meta-hydroxyphenyl)porphyrin), AlPcS₄(aluminium phthalocyanine tetrasulphonate), ZnET2 (zinc aetio-purpurin),and Bchla (bacterio-chlorophyll .alpha.).

In one embodiment, the immunoadjuvant composition is formulated as asolution or suspension. The solution or suspension can include, forinstance, about 0.25% by weight of a chromophore and about 1% by weightof the glycated chitosan.

According to another preferred embodiment, the present inventionprovides a composition for use in conditioning a neoplasm for tandemphotophysical and immunological treatment, comprising an immunoadjuvant,wherein the immunoadjuvant is conjugated to a tumor specific antigen,and wherein the immunoadjuvant is glycated chitosan.

According to still another embodiment, the present invention provides acomposition for use in conditioning a neoplasm for tandem photophysicaland immunological treatment, comprising a combination of a chromophoreand an immunoadjuvant, wherein the chromophore and the immunoadjuvantare conjugated to a tumor specific antigen, and wherein theimmunoadjuvant is glycated chitosan.

According to another preferred embodiment, the present inventionprovides a composition for use in conditioning a neoplasm for tandemphotophysical and immunological treatment, comprising an immunoadjuvant,wherein the immunoadjuvant is conjugated to a tumor specific antibody,and wherein the immunoadjuvant is glycated chitosan. The immunoadjuvantcan, in certain instances, consist essentially of glycated chitosan. Theglycated chitosan can also further include a carbonyl reactive group.

According to another embodiment, the present invention provides acomposition for use in conditioning a neoplasm for tandem photophysicaland immunological treatment, comprising a combination of a chromophoreand an immunoadjuvant, wherein the chromophore and the immunoadjuvantare conjugated to a tumor specific antibody, and wherein theimmunoadjuvant is glycated chitosan. The immunoadjuvant can, in certaininstances, consist essentially of glycated chitosan. The glycatedchitosan can also further include a carbonyl reactive group.

The present invention thus provides injectable formulations forconditioning a neoplasm for tandem photophysical and immunologicaltreatment, that in certain instances may include a combination of, or amixture of, a chromophore and an immunoadjuvant, wherein theimmunoadjuvant is glycated chitosan.

A composition may furthermore be prepared for use in conditioning aneoplasm for tandem photophysical and immunological treatment,comprising an immunoadjuvant, wherein the immunoadjuvant is conjugatedto a tumor specific antigen, and wherein the immunoadjuvant isviscoelastic glycated chitosan with a molecular weight (MW) of betweenabout 100 kDa and about 1000 kDa; and more preferably, between about 100kDa and about 300 kDa. Specifically, in a preferred embodiment themolecular weight is less than 500,000 Daltons.

A composition may also be prepared for use in conditioning a neoplasmfor tandem photophysical and immunological treatment, comprising acombination of a chromophore and an immunoadjuvant, wherein thechromophore and the immunoadjuvant are conjugated to a tumor specificantigen, and wherein the immunoadjuvant is viscoelastic glycatedchitosan with a molecular weight (MW) of between about 100 kDa and about1000 kDa; and more preferably, between about 100 kDa and about 300 kDa.Specifically, in a preferred embodiment the molecular weight is lessthan 500,000 Daltons.

Furthermore, an injectable solution may be prepared for conditioning aneoplasm for tandem photophysical and immunological treatment comprisingan immunoadjuvant wherein the immunoadjuvant is viscoelastic glycatedchitosan with a molecular weight (MW) of between about 100 KDa and about1000 kDa; and more preferably, between about 100 kDa and about 300 kDa.Specifically, in a preferred embodiment the molecular weight is lessthan 500,000 Daltons.

An injectable solution may also be prepared for conditioning a neoplasmfor tandem photophysical and immunological treatment comprising amixture of a chromophore and an immunoadjuvant wherein theimmunoadjuvant is viscoelastic glycated chitosan with a molecular weight(MW) of between about 100 kDa and about 1000 kDa; and more preferably,between about 100 kDa and about 300 kDa. Specifically, in a preferredembodiment the molecular weight is less than 500.000 Daltons.

In one example, the viscoelastic glycated chitosan compositions of thepresent invention is used as an immunoadjuvant in a novel cancertreatment. Photothermal and immunological therapies are combined byirradiating the neoplasm directly to the tumor without the use of achromophore, and subsequently introducing the chitosan-derivedimmunoadjuvant into or around the irradiated neoplasm. Following theapplication of a laser with irradiance sufficient to induce neoplasticcellular destruction, cell-mediated and humoral immune responses to theneoplastic antigens thus released are stimulated (enhanced) by theimmunoadjuvant component.

In another example, photodynamic and immunological therapies arecombined by introducing both a chromophore and a chitosan-derivedimmunoadjuvant (also called immuno-modulator or immunopotentiator) intoa neoplasm. Upon application of a laser with irradiance sufficient toinduce neoplastic cellular destruction, cell-mediated and humoral immuneresponses to the neoplastic antigens thus released are stimulated(enhanced) by the immunoadjuvant component.

The chromophore and immunoadjuvant may be combined into a solution forinjection into the center of the tumor mass or injected separately intothe tumor mass. It should be recognized however that other methods maybe sufficient for localizing the chromophore and immunoadjuvant in thetumor site. One such alternative delivery means is conjugation of thechromophore or immunoadjuvant or both to a tissue specific antibody ortissue specific antigen, such that delivery to the tumor site isenhanced. Any one method, or a combination of varying methods, oflocalizing the chromophore and immunoadjuvant in the tumor site isacceptable so long as the delivery mechanism insures sufficientconcentration of the components in the neoplasm.

According to another embodiment, a method for treating a neoplasm in ahuman or other animal host, comprises: (a) selecting an immunoadjuvant,wherein the immunoadjuvant comprises viscoelastic glycated chitosan; (b)irradiating the conditioned neoplasm whereby neoplastic cellulardestruction of the conditioned neoplasm is induced producing fragmentedneoplastic tissue and cellular molecules; and (c) introducing theimmunoadjuvant into or around the neoplasm, which stimulates theself-immunological defense system of the host to process the fragmentedneoplastic tissue and cellular molecules, such as tumor antigens, andthus create an immunity against neoplastic cellular multiplication.

According to yet another embodiment, a method for treating a neoplasm ina human or other animal host, comprises: (a) selecting a chromophore andan immunoadjuvant, wherein the immunoadjuvant comprises viscoelasticglycated chitosan; (b) introducing the chromophore and theimmunoadjuvant into the neoplasm to obtain a conditioned neoplasm; and(c) irradiating the conditioned neoplasm whereby neoplastic cellulardestruction of the conditioned neoplasm is induced producing fragmentedneoplastic tissue and cellular molecules in the presence of theimmunoadjuvant which stimulates the self-immunological defense system ofthe host against neoplastic cellular multiplication.

In yet another embodiment, a method of producing tumor specificantibodies in a tumor-bearing host, includes irradiating a tumor with alaser of a wavelength in the visible, near-infrared or infrared range,to a degree sufficient to induce neoplastic cellular destruction andgenerating fragmented neoplastic tissue and cellular molecules, followedby the introduction of an immunoadjuvant into or around a neoplasm bymeans of injection so that the host's immune system is stimulated tointeract with and process fragmented neoplastic tissue and cellularmolecules, upon which a systemic anti-tumor response is induced.

In another embodiment, a method of producing tumor specific antibodiesin a tumor-bearing host, includes simultaneously introducing achromophore and an immunoadjuvant into a neoplasm by intratumorinjection to obtain a conditioned neoplasm, the chromophore beingsuitable to generate thermal energy upon activation in the near-infraredor infrared wavelength range; and activating the chromophore with alaser of a wavelength in the near-infrared or infrared range to a degreesufficient to activate the chromophore to produce a photothermalreaction inducing neoplastic cellular destruction and generatingfragmented neoplastic tissue and cellular molecules.

An exemplary method of photophysically destroying a neoplasm andconcurrently generating an in situ autologous vaccine in a tumor-bearinghost, includes: (a) selecting an immunoadjuvant; (b) irradiating theneoplasm with a laser of a wavelength in the visible, near-infrared orinfrared range, at a power and for a duration sufficient to produce aphotothermal reaction inducing neoplastic cellular destruction andgenerating fragmented neoplastic tissue and cellular molecules; (c)forming the in situ vaccine by introducing the immunoadjuvant into theneoplasm by intratumor injection wherein the in situ vaccine comprisesan amalgam of the fragmented tissue and cellular molecules and theimmunoadjuvant; and (d) stimulating the self-immunological defensesystem against neoplastic cellular multiplication by having the vaccinepresented locally to induce an anti-tumor response systemically withinthe host.

Another exemplary method of photophysically destroying a neoplasm andconcurrently generating an in situ autologous vaccine in a tumor-bearinghost, includes: (a) selecting a chromophore and an immunoadjuvant, thechromophore being suitable to generate thermal energy upon activation inthe near-infrared or infrared wavelength range; (b) introducing thechromophore into the neoplasm by intratumor injection; (c) irradiatingthe neoplasm with a laser of a wavelength in the visible, near-infraredor infrared range, at a power and for a duration sufficient to activatethe chromophore to produce a photothermal reaction inducing neoplasticcellular destruction and generating fragmented neoplastic tissue andcellular molecules; (d) forming the in situ vaccine by introducing theimmunoadjuvant into the neoplasm by intratumor injection wherein the insitu vaccine comprising an amalgam of the fragmented tissue and cellularmolecules and the immunoadjuvant; and (e) stimulating theself-immunological defense system against neoplastic cellularmultiplication by having the vaccine presented locally to induce ananti-tumor response systemically within the host.

Yet another exemplary method of photophysically destroying a neoplasmand concurrently generating an in situ autologous vaccine in atumor-bearing host, includes: (a) selecting a chromophore and animmunoadjuvant, the chromophore being suitable to generate thermalenergy upon activation in the near-infrared or infrared wavelengthrange; (b) simultaneously or separately introducing the chromophore andthe immunoadjuvant into the neoplasm by intratumor injection to obtain aconditioned neoplasm; (c) forming the in situ vaccine by irradiating theconditioned neoplasm with a laser of a wavelength in the near-infraredor infrared range at a power and for a duration sufficient to activatethe chromophore to produce a photothermal reaction inducing neoplasticcellular destruction and generating fragmented neoplastic tissue andcellular molecules, wherein the in situ vaccine comprising an amalgam ofthe fragmented tissue and cellular molecules and the immunoadjuvant; and(d) stimulating the self-immunological defense system against neoplasticcellular multiplication by having the vaccine presented locally and byallowing the vaccine to be dispersed systemically within the host.

As described elsewhere herein, the method can further includeconjugating the immunoadjuvant to a tumor specific antibody, therebyforming a conjugate, and administering the conjugate to the host.Alternatively, the method can further include conjugating theimmunoadjuvant to a tumor specific antigen, thereby forming a conjugate,and administering the conjugate to the host. Any number of suitablechromophores can be used, for instance, indocyanine green, DHE, m-THPP,AlPcS₄, ZnET2, and Bchla.

Furthermore, the method can include conjugating a combination of thechromophore and the immunoadjuvant to a tumor specific antibody, therebyforming a conjugate, and administering the conjugate to the host.Alternatively, the method can further include conjugating thechromophore and the immunoadjuvant to a tumor specific antigen, therebyforming a conjugate, and administering the conjugate to the host. Anynumber of suitable chromophores can be used, for instance, indocyaninegreen, DHE, m-THPP, AlPcS₄. ZnET2, and Bchla.

The preparations and formulations of the present invention, includingthe viscoelastic glycated chitosan (GC) preparations, can also be usedin conjunction with photodynamic therapy (PDT). Photosensitizingcompounds show a photochemical reaction when exposed to light.Photodynamic therapy (PDT) uses such photosensitizing compounds andlasers to produce tumor necrosis. Treatment of solid tumors by PDTusually involves the systemic administration of tumor localizingphotosensitizing compounds and their subsequent activation by laser.Upon absorbing light of the appropriate wavelength, the sensitizer isconverted from a stable atomic structure to an excited state.Cytotoxicity and eventual tumor destruction are mediated by theinteraction between the sensitizer and molecular oxygen within thetreated tissue to generate cytotoxic singlet oxygen.

Two good general references pertaining to PDT, biomedical lasers andphotosensitizing compounds, including light delivery and dosageparameters, are Photosensitizing Compounds: Their Chemistry, Biology andClinical Use, published in 1989 by John Wiley and Sons Ltd., Chichester,U.K., ISBN 0 471 92308 7, and Photodynamic Therapy and BiomedicalLasers: Proceedings of the International Conference on PhotodynamicTherapy and Medical Laser Applications, Milan, 24-27 Jun. 1992,published by Elsevier Science Publishers B.V., Amsterdam, TheNetherlands, ISBN 0 444 81430 2, both incorporated herein by reference.

United States patents related to PDT include U.S. Pat. Nos. 5,095,030and 5,283,225 to Levy et al.; U.S. Pat. No. 5,314,905 to Pandey et al.;U.S. Pat. No. 5,214,038 to Allison et al; and U.S. Pat. No. 5,258,453 toKopecek et al., all of which are incorporated herein by reference. TheLevy patents disclose the use of photosensitizers affected by awavelength of between 670-780 nm conjugated to tumor specificantibodies, such as receptor-specific ligands, immunoglobulins orimmunospecific portions of immunoglobulins. The Pandey patents aredirected to pyropheophorbide compounds for use in standard photodynamictherapy. Pandey also discloses conjugating his compositions with ligandsand antibodies. The Allison patent is similar to the Levy patents inthat green porphyrins are conjugated to lipocomplexes to increase thespecificity of the porphyrin compounds for the targeted tumor cells. TheKopecek patent also discloses compositions for treating canceroustissues. These compositions consist of two drugs, an anti-cancer drugand a photoactivatable drug, attached to a copolymeric carrier. Thecompositions enter targeted cells by pinocytosis. The anti-cancer drugacts after the targeted cell has been invaded. After a period of time, alight source is used to activate the photosensitized substituent.

Further Application for Tumor Immunotherapy

The preparations and formulations of the present invention, includingthe viscoelastic glycated chitosan (GC) preparations, can be used, e.g.,as immunoadjuvants, in the context of tumor immunotherapy.

The major functions of the immune system are to develop the concept of“self” and eliminate what is “nonself”. Although microorganisms are theprincipal nonself entities encountered every day, the immune system alsoworks to eliminate neoplasms and transplants.

There are several distinct types of immunity. Nonspecific, or innate,immunity refers to the inherent resistance manifested by a species thathas not been immunized (sensitized or allergized) by previous infectionor vaccination. Its major cellular component is the phagocytic system,whose function is to ingest and digest invading microorganisms.Phagocytes include neutrophils and monocytes in the blood andmacrophages in the tissues. Complement proteins are the major solublecomponent of nonspecific immunity. Acute phase reactants and cytokines,such as interferon, are also part of innate immunity.

Specific immunity is an immune status in which there is an alteredreactivity directed solely against the antigenic determinants(infectious agent or other) that stimulated it. It is sometimes referredto as acquired immunity. It may be active and specific, as a result ofnaturally acquired (apparent or unapparent) infection or intentionalvaccination; or it may be passive, being acquired from a transfer ofantibodies from another person or animal. Specific immunity has thehallmarks of learning, adaptability, and memory. The cellular componentis the lymphocyte (e.g., T-cells, B-cells, natural killer (NK) cells),and immunoglobulins are the soluble component.

The action of T-cells and NK-cells in recognizing and destroyingparasitized or foreign cells is termed cell-mediated Immunity. Incontradistinction to cell-mediated immunity, humoral immunity isassociated with circulating antibodies produced, after a complexrecognition process, by B-cells.

As regards tumor immunology, the importance of lymphoid cells in tumorimmunity has been repeatedly shown. A cell-mediated host response totumors includes the concept of immunological surveillance, by whichcellular mechanisms associated with cell-mediated immunity destroy newlytransformed tumor cells after recognizing tumor-associated antigens(antigens associated with tumor cells that are not apparent on normalcells). This is analogous to the process of rejection of transplantedtissues from a nonidentical donor. In humans, the growth of tumornodules has been inhibited in vivo by mixing suspensions of a patient'speripheral blood lymphocytes and of tumor cells, suggesting acell-mediated reaction to the tumor. In vitro studies have shown thatlymphoid cells from patients with certain neoplasms show cytotoxicityagainst corresponding human tumor cells in culture. These cytotoxiccells, which are generally T-cells, have been found with neuroblastoma,malignant melanomas, sarcomas, and carcinomas of the colon, breast,cervix, endometrium, ovary, testis, nasopharynx, and kidney. Macrophagesmay also be involved in the cell-mediated host's response to tumors whenin the presence of tumor-associated antigens, lymphokines or interferon.

Humoral antibodies that react with tumor cells in vitro have beenproduced in response to a variety of animal tumors induced by chemicalcarcinogens or viruses. Hydridoma technology in vitro permits thedetection and production of monoclonal antitumor antibodies directedagainst a variety of animal and human neoplasms. Antibody-mediatedprotection against tumor growth in vivo, however, has been demonstrableonly in certain animal leukemias and lymphomas. By contrast, lymphoidcell-mediated protection in vivo occurs in a broad variety of animaltumor systems.

Immunotherapy for cancer is best thought of as part of a broadersubject, namely biologic therapy, or the administration ofbiologic-response modifiers. These agents act through one or more of avariety of mechanisms (1) to stimulate the host's antitumor response byincreasing the number of effector cells or producing one or more solublemediators; (2) to serve as an effector or mediator; (3) to decrease hostsuppressor mechanisms; (4) to alter tumor cells to increase theirimmunogenicity or make them more likely to be damaged by immunologicalprocesses; or (5) to improve the host's tolerance to cytotoxics orradiation therapy. Heretofore the focus of cell-mediated tumorimmunotherapy has been on reinfusion of the patient's lymphocytes afterexpansion in vitro by exposure to interleukin-2. One variation includesisolating and expanding populations of lymphocytes that have infiltratedtumors in vivo, so-called tumor-infiltrating lymphocytes. Another is theconcurrent use of interferon, which is thought to enhance the expressionof histocompatibility antigens and tumor-associated antigens on tumorcells, thereby augmenting the killing of tumor cells by the infusedeffector cells.

Humoral therapy has long concentrated on the use of antitumor antibodiesas a form of passive immunotherapy, in contrast to active stimulation ofthe host's own immune system. Another variation is the conjugation ofmonoclonal antitumor antibodies with toxins, such as ricin ordiphtheria, or with radioisotopes, so the antibodies will deliver thesetoxic agents specifically to the tumor cells. Active immunization with ahost's own tumor cells, after irradiation, neuraminidase treatment,hapten conjugation, or hybridization has also been tried. Clinicalimprovement has been seen in a minority of patients so treated. Tumorcells from others have been used after their irradiation in conjunctionwith adjuvants in acute lymphoblastic leukemia and acute myeloblasticleukemia after remission. Prolongation of remissions or improvedreinduction rates have been reported in some series, but not in most.Interferons, tumor necrosis factor and lymphotoxins have also been usedto affect Immunologically mediated mechanisms. A recent approach, usingboth cellular and humoral mechanisms, is the development of“heterocross-linked antibodies,” including one antibody reacting withthe tumor cell linked to a second antibody reacting with a cytotoxiceffector cell, making the latter more specifically targeted to thetumor. Host immune cell infiltration into a PDT treated murine tumor hasbeen reported.

Combined PDT and Immunotherapy

In accordance with the present invention, it is desirable to utilizeglycated chitosan (GC) preparations having a suitable viscosity thatenables their use as an injectable material in additional applications,such as combined photodynamic cancer therapy (PDT) and tumorimmunotherapy methods.

The potential for combining PDT with immunotherapy was explored byKorbelik, Krosl, Dougherty and Chaplin. See Photodynamic Therapy andBiomedical Lasers, supra, at pp. 518-520. In their study, theyinvestigated a possibility of amplification of an immune reaction to PDTand its direction towards more pervasive destruction of treated tumors.The tumor, a squamous cell carcinoma SCCVII, was grown on female C3Hmice. An immunoactivating agent SPG (a high molecular weight B-glucanthat stimulates macrophages and lymphoid cells to become much moreresponsive to stimuli from cytokines and other immune signals) wasadministered intramuscularly in 7 daily doses either ending one daybefore PDT or commencing immediately after PDT. Photofrin based PDT wasemployed; photofrin having been administered intravenously 24 hoursbefore the light treatment. The SPG immunotherapy was shown to enhancethe direct killing effect of the PDT. The indirect killing effect (seenas a decrease in survival of tumor cells left in situ) was, however,much more pronounced in tumors of animal not receiving SPG. Thedifference in the effectiveness of SPG immunotherapy when performedbefore and after PDT suggested that maximal interaction is achieved whenimmune activation peaks at the time of the light delivery or immediatelythereafter. With SPG starting after PDT (and attaining an optimal immuneactivation 5-7 days later), it is evidently too late for a beneficialreaction.

In another study the use of PDT to potentiate the effect of bioreactivedrugs that are cytotoxic under hypoxic conditions was investigated. SeePhotodynamic Therapy and Biomedical Lasers, supra, at pp. 698-701. Itwas found that the antitumor activity of such drugs could be enhanced invivo when they were used in combination with treatments that increasetumor hypoxia.

Cancer Treatment by Photodynamic Therapy, in Combination with anImmunoadjuvant

In accordance with the present invention, it is desirable to utilizeglycated chitosan (GC) preparations having a suitable viscosity asinjectable materials for use in the treatment of cancer. This can beachieved in any suitable manner, for instance, in conjunction withapplications such as combined photothermal or photodynamic cancertherapy (PDT) and tumor immunotherapy methods. The term cancer, as usedherein, is a general term that is intended to include any of a number ofvarious types of malignant neoplasms, most of which invade surroundingtissues, may metastasize to several sites, and are likely to recur afterattempted removal and to cause death of the patient unless adequatelytreated. A neoplasm, as used herein, refers to an abnormal tissue thatgrows by cellular proliferation more rapidly than normal. It continuesto grow even after the stimulus that initiated its growth dissipates.Neoplasms show a partial or complete lack of structural organization andfunctional coordination with the normal tissue and usually form adistinct mass which may be either benign or malignant.

In accordance with the present invention, certain examples of cancersthat may be treated with glycated chitosan (GC) preparations having asuitable viscosity as injectable materials include, but are not limitedto, those of the cervix, breast, bladder, colon, prostate, larynx,endometrium, ovary, oral cavity, kidney, testis (nonsemino-matous) andlung (non-small cell).

Moreover, in accordance with the present invention, treatment may alsobe administered in a suitable manner in conjunction with other types ofcancer treatment, for instance, radiation treatment. Radiation plays akey role, for example, in the remediation of Hodgkin's disease, nodularand diffuse non-Hodgkin's lymphomas, squamous call carcinoma of the headand neck, mediastinal germ-cell tumors, seminoma, prostate cancer, earlystage breast cancer, early stage non-small cell lung cancer, andmedulloblastoma. Radiation can also be used as palliative therapy inprostate cancer and breast cancer when bone metastases are present, inmultiple myeloma, advanced stage lung and esophagopharyngeal cancer,gastric cancer, and sarcomas, and in brain metastases. Cancers that maybe treated include, for instance, Hodgkin's disease, early-stagenon-Hodgkin's lymphomas, cancers of the testis (seminormal), prostate,larynx, cervix, and, to a lesser extent, cancers of the nasopharynx,nasal sinuses, breast, esophagus, and lung.

Treatment may also be administered in a suitable manner in conjunctionwith other types of antineoplastic drugs. Antineoplastic drugs includethose that prevent cell division (mitosis), development, maturation, orspread of neoplastic cells. The ideal antineoplastic drug would destroycancer cells without adverse effects or toxicities on normal cells, butno such drug exists. Despite the narrow therapeutic index of many drugs,however, treatment and even cure are possible in some patients. Certainstages of choriocarcinoma, Hodgkin's disease, diffuse large celllymphoma, Burkitt's lymphoma and leukemia have been found to besusceptible to antineoplastics, as have been cancers of the testis(nonseminomatous) and lung (small cell). Common classes ofantineoplastic drugs include, but are not limited to, alkylating agents,antimetabolites, plant alkaloids, antibiotics, nitrosoureas, inorganicions, enzymes, and hormones.

In Situ Autologous Cancer Vaccines, Such as Laser-Assisted Immunotherapy

The chitosan-derived compositions and, in particular, the viscoelasticglycated chitosan preparations of the present invention, are effectivein treating neoplasms and other medical disorders. Additional uses ofglycated chitosan, alone or in combination with other drugs, include useas an immunostimulant in the treatment of immuno-compromised patientsincluding but not limited to cancer and acquired immunodeficiencysyndrome.

The chitosan-derived compositions of the present invention are thususeful in a myriad of applications, including for instance as animmunoadjuvant or as a component of an immunoadjuvant, as described indetail herein. Notwithstanding other uses, a principal use of thechitosan-derived compositions is as an immunoadjuvant in connection within situ autologous cancer vaccines (inCVAX), such as laser-assistedimmunotherapy (LIT), and it is in this context that the chitosan-derivedcompositions are described in detail herein.

As described further herein, additional embodiments of the presentinvention are directed to uses of the glycated chitosan preparations ofthe present invention as immunoadjuvants in conjunction with inCVAX ingeneral, and LIT in particular, for cancer treatment. Laser-assistedimmunotherapy utilizing the present invention preferably encompassesintroducing into or around a neoplasm an immunoadjuvant comprisingviscoelastic chitosan-derived compositions following photothermalirradiation of the same tumor. The photothermal action is performed atan irradiance sufficient to induce neoplastic cellular destruction,which can be performed with or without intratumoral injection of, or byother means delivered, a chromophore, and combined with injection of, orby other means delivered, the viscoelastic glycated chitosanpreparations of the present invention, cell-mediated and humoralanti-tumor immune responses are induced.

In preferred embodiments, improved LIT is provided wherein theimprovement comprises the use of the herein-described injectableviscoelastic glycated chitosan preparations of the present invention.The present invention also contemplates methods of in vivo activation ofspecific components of the immune system in conjunction with inCVAX ingeneral, or LIT in particular, comprising treatment with a viscoelasticglycated chitosan preparation.

As described further herein, it has been determined that LIT provides anin situ autologous cancer vaccine (inCVAX) that overcomes limitations ofcurrent immunotherapies and cancer vaccines. In general, the twoprinciples underlying LIT are (1) local heating of the primary tumorwith a laser to devitalize the tumor and liberate tumor antigens, and(2) local injection of a potent and nontoxic immunoadjuvant comprisingglycated chitosan (GC), which interacts with liberated tumor antigens toinduce an immune response against the cancer. Thus, LIT effectivelyfunctions as an in situ autologous cancer vaccine that uses whole tumorcells as the sources of tumor antigens from each individual patientwithout pre-selection of tumor antigens or ex vivo preparation.

In accordance with the present invention, another advantage of using theherein-described injectable viscoelastic glycated chitosan preparationsof the present invention, in conjunction with LIT, is that by using thisLIT approach, there is activation of dendritic cells (DC), andsubsequently exposure of the activated DC to tumor antigens in vivo. LITthus represents an advantageous approach to other whole-cell cancervaccinations, by eliminating the need of ex vivo preparations, and byusing LIT in conjunction with the viscoelastic glycated chitosanpreparations as immunoadjuvants.

One exemplary formulation of a glycated chitosan preparation wasmanufactured under the name PROTECTIN. It has been observed thatPROTECTIN in conjunction with LIT stimulates the immune system andinduces tumor-specific immunity by 1) activating dendritic cells, 2)increasing the interaction between tumor cells and dendritic cells, and3) increasing the tumor antigen presentation to the immune system.

Other viscoelastic glycated chitosan preparations of the presentinvention also function to stimulate the immune system and inducetumor-specific immunity by 1) activating dendritic cells, 2) increasingthe interaction between tumor cells and dendritic cells, and 3)increasing the tumor antigen presentation to the immune system.

Thus, in accordance with a preferred embodiment of the invention,formulations of viscoelastic glycated chitosan activate one or morecomponents of the immune system, mediating desired therapeutic effects.

As described further herein, certain components of the immune systemthat are activated include components of nonspecific, or innate,immunity, namely the phagocytic system including neutrophils andmonocytes in the blood and macrophages in the tissues; complementproteins, the major soluble component of nonspecific immunity; and acutephase reactants and cytokines, such as interferon, also part of innateimmunity. There are many different components of specific immunity, forexample, the lymphocyte (e.g., T-cells, B-cells, natural killer (NK)cells), and immunoglobulins. The glycated chitosan formulations of theinvention also interact with lymphoid cells to promote tumor immunity.Macrophages may also be involved in the cell-mediated hosts response totumors when in the presence of tumor-associated antigens, lymphokines orinterferon.

Specific components of the immune system are activated after“photothermal” treatment. When photothermal destruction occurs, thefragmented tissue and cellular molecules are disbursed within the hostin the presence of the immunologically potentiating material, such aschitosan. In effect, an in situ vaccine is formed. This mixture ofmaterials then circulates in the host and is detected by theimmunological surveillance system. There follows an immediatemobilization of cell-mediated immunity which encompasses NK-cells andrecruited killer T-cells. These cells migrate to the sites of similarantigens or chemicals. In time, the cell-mediated immunity shifts to ahumoral immunity with the production of cytotoxic antibodies. Theseantibodies freely circulate about the body and attach to cells andmaterials for which they have been encoded. If this attachment occurs inthe presence of complement factors, the result is cellular death.

The injectable viscoelastic glycated chitosan preparations of thepresent invention have unexpected utility in “in situ cancer vaccines”,which are based on an in situ activation of antigen-presenting cells(e.g., dendritic cells and macrophages), and the subsequent exposure oftumor antigens to the antigen-presenting cells. The injectableviscoelastic glycated chitosan preparations of the present inventionalso activate other cellular mediators including, but not limited to,tumor necrosis factor (e.g., TNFa) and nitric oxide which contribute tothe therapeutic effects.

Another advantage of using the herein-described injectable viscoelasticglycated chitosan preparations of the present invention, in conjunctionwith LIT, is that by using this approach, this method independentlytriggers the immune response in each individual, and it does not dependupon cross reactivity in the expression of tumor-specific antigenbetween hosts (as is required in conventional antibody immunotherapy andvaccination). Histochemical studies have revealed that sera fromLIT-cured tumor-bearing rats contained antibodies that bound to theplasma membrane of both living and preserved tumor cells. Western blotanalysis of tumor cell proteins using sera (from rats successfullytreated by LIT) as the source of primary antibodies showed distinctbands, indicating induction of tumor-selective antibodies. It was alsoshown that successfully treated rats could acquire long-term resistanceto tumor re-challenge, and adoptive immunity could be transferred usingspleen cells from successfully treated rats, indicating tumor-specificimmunity.

Thus, using the herein-described injectable viscoelastic glycatedchitosan preparations of the present invention, there are severaladvantages that meet critical needs in providing effective cancertreatment. This is particularly advantageous for cancer patients, sincethe present invention also provides surprisingly and unexpectedlybeneficial preparations that are easy to administer by injection, andtherefore increase compliance and provide effective treatmentalternatives to conventional approaches that do not provide (1)effective, (2) nontoxic, and (3) practical treatments for late-stagemetastatic cancer. A critical issue in breast cancer therapy is that notall patients are treatable with current, conventional methodologies andthose diagnosed at late stages have a poor prognosis, with even fewervalid options for treatment. And, while there have been many advancesand developments in breast cancer treatment in recent years, crucialproblems remain. The injectable viscoelastic glycated chitosanpreparations of the present invention, as described herein, provideseveral advantages that meet critical needs in providing effectivecancer treatment.

LIT has been shown to induce maturation of dendritic cells (assessed byCD80 expression), enhance T-cell proliferation, increase IFN-γ secretionand increase HSP70 expression. Furthermore, the combined effects of LIT(for instance, tumor heating with a laser and injection of glycatedchitosan preparations in accordance with the present invention) has beenshown to induce tumor-specific immunity, with an infiltration oftumor-specific cytotoxic CD4 and CD8 cells into the tumors following thetreatment.

As described in further detail herein, LIT thus provides numerousadvantages including, but not limited to:

-   -   Eliminates treated primary tumors    -   Eliminates untreated metastases    -   Induces long-term immunity and survival    -   Creates resistance to tumor rechallenges    -   Is non-toxic and safe to use in humans at therapeutic doses

In accordance with one aspect of the invention, a neoplasm, such as amalignant tumor, is irradiated with visible, near-infrared or infraredlight with a power and a duration sufficient to elevate the temperatureof the neoplasm to a level that induces neoplastic cellular destructionand stimulates the self-immunological defense system against neoplasticcellular multiplication. To facilitate the heating of the tumor, achromophore with absorption peaks corresponding to the wavelength of theapplied light, may be injected prior to applying the light treatment.Following the light irradiation, a viscoelastic glycatedchitosan-derived immunoadjuvant is administered, for example byinjection, into the tumor or the tissue immediately surrounding thetumor.

In accordance with another aspect of the invention, a solution ofindocyanine green (ICG) and glycated chitosan is prepared at aconcentration of 0.1 to 2% of ICG to chitosan. The solution is injectedinto the neoplasm, and the neoplasm is then irradiated using a laserhaving a power of about 5 watts and a wavelength of radiation capable ofreadily penetrating normal cellular tissues without significantdisruption. The irradiation continues for a duration of from about oneto about ten minutes, which is sufficient to elevate the temperature ofthe neoplasm to a level that induces neoplastic cellular destruction andstimulates cell-mediated and humoral immune responses.

As described further herein, the present invention has severaladvantages over other conventional and unconventional treatmentmodalities. The combination of tumor destruction and immune-stimulationadjuvant is the key. The most significant advantage is combined acuteand chronic tumor destruction. The acute tumor loss is caused byphotovaporization, photoablation or thermal killing of the neoplastictissue, on a large and controlled scale, in the immediate area, reducingthe tumor burden and hence the base of multiplication so that theself-defense system can fight a weaker “enemy”. When local tumordestruction occurs, the fragmented tissue and cellular molecules arelocally disbursed within the host in the presence of the immunologicallypotentiating material, such as glycated chitosan. In effect, an in situvaccine is formed. There follows an immediate mobilization ofcell-mediated immunity which encompasses NK-cells and recruited killerT-cells. These cells migrate to the sites of similar antigens orchemicals. In time, the cell-mediated immunity shifts to a humoralimmunity with the production of cytotoxic antibodies. These antibodiesfreely circulate about the body and attach to cells and materials forwhich they have been encoded. If this attachment occurs in the presenceof complement factors, the result is cellular death. The time frames forthese two immunological modes of action are 0 to 2 weeks for thecell-mediated response, while the humoral arm matures at approximately30 days and should persist for long periods, up to the life span of thehost.

In summary, long-term survival with total cancer eradication can beachieved by using the viscoelastic glycated chitosan preparations of thepresent invention. It is a combined result of reduced tumor burden dueto ablative (for example photothermal) interactions and an enhancedimmune system response due to the presence of glycated chitosan or otherimmunomodulators.

According to other embodiments, the glycated chitosan preparations ofthe present invention may also be used for antimicrobial and/orhemostatic applications. Thus the glycated chitosan (GC) preparationscan be formulated, for instance, as an antimicrobial hemostatic spray,wherein the GC formulation has a viscosity and exhibits rheologicalproperties that enable it to be sprayed from conventional containers.Moreover, GC can be included in other formulations provided that it isapplied in antimicrobial and/or hemostatic effective concentrations andwith viscosities/rheological properties that enable its ability to bedispensed from containers suitable for the purpose.

The present invention is further illustrated by the following examples.These examples are provided by way of illustration and are not intendedin any way to limit the scope of the invention. The examples shouldtherefore not be construed as limitations on the scope of the invention,but rather should be viewed as exemplifications of preferred embodimentsthereof. Many other variations are possible.

EXAMPLES Example 1

Exemplary Process for the Preparation of Glycated Chitosan (GC)

Glycated chitosan is obtained by reacting chitosan with a monosaccharideand/or oligosaccharide, preferably in the presence of an acidifyingagent, for a time sufficient to accomplish Schiff base formation betweenthe carbonyl group of the sugar and the primary amino groups of chitosan(also referred to herein as glycation of the amino group) to apredetermined degree whereby a predetermined percent (%) glycation ofthe chitosan polymer is achieved. This is followed by stabilization byreduction of Schiff bases and of their rearranged derivatives (Amadoriproducts). NMR tracings are used to verify the bonding of themonosaccharides and/or oligosaccharides to the chitosan polymer, whereaschemical measurement of remaining free amino groups, such as via aninhydrine reaction, is used to assess the degree of glycation.

Example 2

Sterile Filtration

While conventional 1500 kDa galactochitosan, described in U.S. Pat. No.5,747,475, is relatively simple to synthesize, the sterilization with,for example a 0.22 micron filter, is impossible without compromising theintegrity of the filter, thus rendering the conventional glycatedchitosan unsuitable for GMP production and human use. In contrast, thenew viscoelastic glycated chitosan described herein has significantadvantages with regard to GMP production and sterile filtration due tounexpected and beneficial physiochemical properties. For example, at amolecular weight (M.W.) of 250,000 Da (250 kDa), sterile filtration witha 0.22 micron filter is highly feasible, with a flow rate of 100 ml/minwithout loss of material during filtration.

Example 3

Viscosity of Glycated Chitosan (GC)

GC preparations of higher molecular weight display higher viscosities(measured in Cp):

kDa of GC Cp 100 0.914 250 7.68 500 20.79 1500 84.7

FIG. 3 shows viscosity (in Cp; y-axis) vs. molecular weight (in kDa;x-axis) in samples of GC with molecular weights ranging from 100 kDa to1,500 kDa. The concentration of GC in solution in this experimentdecreased with increasing molecular weight, ranging from 0.6% (100 kDa)to 0.11% (1,500 kDa).

Very surprisingly, it was found viscosity increases linearly withincreasing molecular weight only if the concentration of GC in thesample is reduced with increasing molecular weight. The table and FIG. 4shows the percent of GC in solution of the samples used in the viscosityexperiment above.

Size kDa Percent GC in Sample 100 0.6 250 0.3 500 0.14 1500 0.11

The results clearly show that 1) GC preparations of higher molecularweight correlate with higher viscosities (measured in Cp), and 2) thecorrelation between viscosity and molecular weight is not linear if theconcentration is kept constant. In other words, the viscosity increasesdisproportionally with increasing molecular weight, which renders thehigher molecular weight glycated chitosans (such as those disclosed inU.S. Pat. No. 5,747,475) unsuitable for injection or sterile filtration.

Viscoelastic glycated chitosan preparations comprising lower molecularweight (i.e. below ˜400 kDa) glycated chitosan thus provide improvedinjectability; these preparations are useful, for instance, for cancertreatments utilizing photodynamic therapy and laser-assistedimmunotherapy to induce neoplastic cellular destruction and to stimulatethe self-immunological defense system against neoplastic cells.

Example 4

Improvement of Manufacturing

in this exemplary study, it was determined that experimental conditionscould be adjusted as needed to improve overall yield during themanufacture of glycated chitosan. It was unexpectedly discovered thatmanufacturing of GOC could be improved by controlling the pH conditions,and thus controlling the percent glycation. Specifically, it wasdetermined that because the half-life of sodium borohydride (NaBH₄) isproportional to pH, meaning that at lower pH the half-life of NaBH₄ isextremely short, and only at higher pH is the NaBH₄ somewhat morestable. It was thus determined that NaBH₄ was not as effective instabilizing the glycated chitosan by reduction of the Schiff bases andAmidori products at lower pH. For instance, when the pH was kept belowfive (pH<5), the half-life of NaBH4 is extremely short, and thus thereduction of the Schiff bases and Amadori products was less efficient,and percent glycation of GC thus went down.

It was determined, however, that with a higher pH, the formulation“gels” and becomes non-newtonian. For instance, when the pH was keptabove six (pH>6), the formulation was observed to gel and thus the batchhad to be discarded. In other words, to achieve the goal of efficient GCmanufacturing, the pH was not kept so high that the formulation would“gel”, but the pH was also not kept so low that the percent glycationwas minimized due to the short half life of NaBH₄.

Example 5

Laser-Assisted Immunotherapy (UT) Treatment in a Human Trial

An investigator-driven breast cancer trial was performed on 10 patientswith advanced breast cancer (5 stage IV, 5 stage III). Most of thepatients had responded poorly, or not at all, to conventionalmodalities, and received at least one Laser-Assisted Immunotherapy (LIT)treatment in which viscoelastic glycated chitosan was used as theimmunoadjuvant. Two (2) patients withdrew prematurely due to unrelatedreasons, leaving 8 evaluable patients. The independent investigatorsacquired IRB and government approvals prior to the trials. Biopsies andmedical imaging (CT scans, etc.) were used for the evaluation of theprimary lesions and metastasis.

The primary efficacy parameter was the best overall response by theinvestigators' assessments using Response Evaluation Criteria in SolidTumors (RECIST). Complete response (CR) was defined as disappearance orlack of qualifying metabolic activity of all target lesions. Partialresponse (PR) was defined as a ≥30% decrease from baseline in activityor in the sum of the longest diameter of target lesions. Progressivedisease (PD) is defined as a ≥20% increase in the sum of the longestdiameter of target lesions or the appearance of 1 or more new lesions.Stable disease (SD) was defined as neither sufficient reduction toqualify for PR nor sufficient increase to qualify for PD.

Of the 8 breast cancer patients available for evaluation, CR wasobserved in 1 patient, PR in 4 patients and SD in 1 patient. In patientsavailable for evaluation, the objective response rate (CR+PR) was 62.5%,and the clinically beneficial response rate (CR+PR+SD) was 75%. PD wasobserved in 2 patients. All local lesions irradiated by laser respondedto LIT. In addition, most of the distant metastases of these patientsresponded to LIT. The diameters and activity of the metastases in lymphnode, lung and liver in several patients decreased dramatically.

Local and systemic toxicity was graded according to National CancerInstitute Common Toxicity Criteria, version 3.0. Laboratory assessmentand physical examinations were performed periodically. Adverse eventswere closely monitored and recorded throughout the study period. LITonly induced local reactions within the treatment area in breast cancerpatients, most of which were related to the thermal effects of thetopical laser treatment. Redness, pain, edema and ulceration of thetreatment area were the common adverse events (AEs). No grade 3 or 4adverse events were observed. In patients who had not received priorradiation therapy the swelling was minor. For the patients who havereceived prior radiation therapy, the swelling was more substantial withlonger duration.

Example 6

Laser-Assisted Immunotherapy with Glycated Chitosan DemonstratesAntitumor Immunity Against 816 Melanoma Tumors in Mice

Female C57BC/6 mice (8 weeks of age; 12 mice/group) were subcutaneouslyinoculated with the B16-F1 melanoma tumor (10⁶ viable tumor cells) intothe back area. The tumors reached treatment size (7 to 8 mm in diameter)around 7 days after implantation. Five treatment groups (12 femalemice/group) were included in the study: an untreated control;laser-assisted immunotherapy treatment control; and laser-assistedimmunotherapy treatment with 0.2 mL of 1% glycated chitosanperitumorally injected 24 h prior, immediately following, or 24 h afterlaser treatment. The 805 nm diode laser was used for laser irradiation,with parameter settings of 2 W for 10 min in duration. The laser wasdirected through an optical fiber with a diffuser lens at the end to thetreatment site and the laser tip was maintained at a distance of 4 mmfrom the skin.

Animal survival was evaluated. Darkening and hardening of the mouse skinat the treatment site was observed after laser treatment. Tumorreoccurrence usually occurred several days after treatment. Thermaltreatment in combination with glycated chitosan application resulted ina significant improvement in animal survival with glycated chitosanadministered 24 h before laser irradiation showing the most significantimprovements (see table below).

Effect of laser and Glycated Chitosan Treatment in B16 MelanomaTumor-Bearing Mice^(n) Long-Term (>90 Days) Treatment Injection*Survival Rate (%)^(b) Untreated Control 0.0 Laser Only 16.7 Laser + 0.2mL 1% GC 24 h 16.7 After Laser Laser + 0.2 mL 1% GC 24 h 25.05Immediately After Laser Laser + 0.2 mL 1% GC 24 h 41.7 Before Laser * =805 nm diode laser with energy (2W, 10 min.) directed through an opticalfiber with a diffuser lens that was maintained at a distance of 4 mmfrom the skin ^(b) = Long-term survival was defined as >90 days afterinoculation without tumor recurrence GC = Glycated chitosan ^(n) = 12female C57BC/6 mice

Example 7

Interstitial Laser-Assisted Immunotherapy in a Metastatic Mammary ModelUsing 805 nm Laser and Glycated Chitosan

A study was conducted to determine the optimal interstitial laser doseand the optimal glycated chitosan dose. Female Wistar Furth rats (5 to 6weeks of age, 100 to 125 g) were subcutaneously injected with thetransplantable, metastatic mammary tumor, DMBA-4, (10⁵ viable tumorcells) into the back area. DMBA-4 tumors, originally induced chemically,are highly metastatic and poorly immunogenic. The tumors metastasizealong the lymphatics and rapidly form multiple metastases at distantsites, killing all the rats 30 to 40 days after tumor implantation. Whenthe primary tumor was 0.2 to 0.5 cm³, the hair overlying the tumor wasdipped and laser-assisted immunotherapy was performed on anesthetizedanimals (2% isofluorane). An 805 nm diode laser was used to delivernear-infrared light for target tumors. Continuous laser power wasdelivered through an optical fiber with an active cylindrical tip. Anactive tip of 1.0 cm was used, with a transparent plastic sheath toprotect the active tip. For the insertion of the active fiber tip,either needle-guided or puncture-assisted insertion methods were used.The intratumoral position of the fiber was verified by a digital camera,which can capture the infrared light from the 805-nm laser. The ratswere observed daily and the tumors were measured twice a week for aperiod of at least 100 days. The criterion for successful treatment wasa 100-day survival after tumor implantation. The optimal interstitiallaser dose was determined by evaluating effects in a control (9 rats, notreatment); interstitial laser powers of 1, 1.5, 2, 2.5, and 3 W/cm² for10 min (14 rats/group); and interstitial laser power of 2 W/cm² for 30min (14 rats/group). The rats in the 3 W at 10 min and 2 W at 30 minappeared to have average survival rates higher than other groups. Theoptimal glycated chitosan dose was determined by evaluating survivalfollowing administration of 0.1, 0.2, 0.4, and 0.6 mL of 1% glycatedchitosan following interstitial laser-assisted immunotherapy at 2.5 Wfor 20 min. A group of rats that received no treatment was included as acontrol. The best survival, at 42%, was observed following a 0.2 mLglycated chitosan dose (see FIG. 5 ).

Example 8

Induced Antitumor Immunity Against DMBA-4 Metastatic Mammary Tumors inRats Using Laser-Assisted Immunotherapy

Female Wistar Furth rats (6 to 7 weeks of age, 110 to 130 g) wereinoculated with the DMBA-4 transplantable, metastatic mammary tumor (10⁵viable tumor cells) into the inguinal area. The primary tumor generallyappeared 7 to 10 days after inoculation and was approximately 1 to 5 gwithin 3 weeks. The tumor metastasized through the lymphatics toinguinal and axillary lymph nodes. Treatment was initiated when theprimary tumor was 0.2 to 0.5 cm³, generally 10 to 15 days afterinoculation. Rats were administered a 0.25% indocyanine green and 1%Glycated Chitosan Solution (0.20 ml) injected directly into the centerof the tumor prior to irradiation. An 805 nm diode laser was used forlaser irradiation, with parameter settings of 2 W for 10 min induration. The laser was directed through an optical fiber to thetreatment site. Following irradiation, animals were housed individuallyand observations and tumor measurements were recorded twice weekly. Ratswhich were successfully treated (cured rats) were rechallengedrepeatedly with the same tumor cells at tumor dose levels of 10⁵ to 10⁷viable tumor cells per rat and animals were observed for 4 months fortumor development. Of the 32 rats treated by laser-assistedimmunotherapy, eight rats were successfully treated and tumor-freefor >120 days following inoculation. In all cured rats, metastasescontinued to develop after treatment, then gradually declined andeventually disappeared without additional treatment. Sevensuccessfully-treated rats were rechallenged up to three times with doselevels ranging from 10⁵ to 10⁷ viable tumor cells per injection. Therewas no primary or metastatic tumor reemergence in any of these animalsand animals survived >120 days, while untreated control rats developedprimary and metastatic tumors and had an average survival of 30 days.

Example 9

Enhancement of Laser Cancer Treatment by a Chitosan-DerivedImmunoadjuvant

The effect of the immunoadjuvant during the laser-assisted immunotherapytreatment was evaluated in rats using four different immunoadjuvants.Female Wistar Furth rats (6 to 8 weeks of age, 150 to 200 g) weresubcutaneously inoculated with the DMBA-4 transplantable, metastaticmammary tumor (10⁵ viable tumor cells) in the inguinal fat pad, 7 to 10days before treatment. The primary tumor generally became palpable in 5to 7 days and the remote inguinal and axillary metastases appeared 15 to20 days after inoculation. The laser-assisted immunotherapy treatmentwas initiated when the primary tumor reached 0.2 to 0.5 cm³. Lasertreatment was generally performed on Day 10. The immunoadjuvantsincluded aqueous 1% Glycated Chitosan Solution (0.2 mL dose; n=48 ratsin two experiments), 50% Complete Freund's Adjuvant (0.2 mL dose; n=33rats), 50% Incomplete Freund's Adjuvant (0.2 mL dose; n=30 rats), andCorynebacterium parvum (C. parvum; 35 μg/rat dose; n=32 rats). Theimmunoadjuvants were mixed with 0.25% indocyanine green and injecteddirectly into the center of the tumor 2 h before irradiation with the805 nm diode laser. Animals were anesthetized prior to irradiation andthe hair overlying the primary tumor was dipped. The laser parameterswere 2 W for 10 min with a 3 mm diameter laser treatment site, resultingin a fluence of 96 J/cm² for a 1 cm diameter tumor. Animals wereindividually housed, observed daily, and tumor burden measurements werecollected twice a week.

3 Data from this study was compared with data from tumor-bearing controlrats (n=38 rats) in several different experiments. All immunoadjuvantshad a statistically significant increase in survival rate compared tocontrol data (p<0.05). The 1% glycated chitosan appeared to be the mosteffective immunoadjuvant with a 29% long-term survival rate (see tablebelow). Statistical significance was observed when the glycated chitosanadjuvant was compared to the C. parvum (p=0.009) and Incomplete Freund'sAdjuvant (p=0.03). Although not significant, a noticeable improvement insurvival was observed when compared to Complete Freund's Adjuvant thathad a comparable cure rate (18%). A relative weak survival rate wasobserved following treatment with the Incomplete Freund's Adjuvant andC. parvum.

Long-term Survival Rates Following treatment with Four DifferentImmunoadjuvants Number of Long-Term Treatment Rats Survival Rate (%)Control  38^(a) 0 Laser + ICG + Glycated Chitosan  48^(b) 29 Laser +ICG + Complete 33 18 Freund's Adjuvant Laser + ICG + Incomplete 30 7Freund's Adjuvant Laser + ICG + C. parvum 32 9 ^(a) = Tumor-bearingcontrol rat data was collected from several control groups in differentstudies ^(b) = Data collecte from two separate experiments ICG =Indocyanine green

Example 10

Enhancement of Photodynamic Therapy by a Chitosan-Derived Immunoadjuvant

To evaluate photodynamic therapy as a the method for direct tumordestruction in combination with glycated chitosan, a combination ofphotofrin- and meso-substituted tetra (meta-hydroxy-phenyl)chlorin-(mTHPC) based photodynamic therapy and glycated chitosaninjection was been studied in the EMT6 mammary sarcoma and Line 1 lungadenocarcinoma mouse models, respectively. In each model, BALB/c micewere subcutaneously inoculated with 10⁶ viable tumor cells into thelower dorsal area. Tumors were treatment size (7 to 8 mm) after 7 days.

In the EMT6 mammary sarcoma model, treatment groups evaluated aredetailed in the table below. Photofrin (Mont-Saint-Hilaire, Quebec,Canada) was prepared in 5% sterile dextrose to a 1 mg/mL concentration.A 5 mg/kg dose of photofrin was intravenously administered 24 h prior toirradiation. Animals were shielded from direct light immediately afterthe photosensitizer injection until 3 days after photodynamic treatment.Mice were restrained anaesthetized in holders exposing their backsduring light treatment. Light (630 nm) was delivered through an 8 mmdiameter liquid light guide. The power density was set at 100 mW/cm²,for a total light dose of 60 J/cm². Immediately after light irradiation,if applicable, animals were administered a peritumoral dose of 0.5 or1.5% glycated chitosan. Animals were observed for tumor emergence every2 days up to 90 days after photodynamic treatment and changes in tumorvolume was determined 3 times a week.

Survival Rates After Photofrin-Based Photodynamic and Glycated ChitosanTreatment in Mice Bearing EMT6 Mammary Tumors Number of Number Long-TermLong-term Treatment of Mice Surviving Mice Survival Rate (%) Control 8 00.0 Non-Thermal Laser 8 0 0.0 Only Non-Thermal Laser + 8 0 0.0 1.5%GC^(a) Non-Thermal Laser + 8 3 37.5 Photofrin Non-Thermal Laser + 8 562.5 Photofrin + 0.5% GC Non-Thermal Laser + 8 6 75.0 Photofrin + 1.5%GC ^(a) = It should be noted that the laser treatment did not result inheating the tumor because the light absorbing agent was not used nd thelaser power was not sufficient to heat the tumor. Therefore, this groupis not representative of the laser-assisted immunotherapy system. GC =Glycated chitosan Laser treatment with a fluence rate of 100 mW/cm² anda total light dose of 60 J/cm², 5 mg/kg photofrin was intravenousyadministered 24 h prior to irradiation. 0.1 mL of 0.5% glycated chitosanwas injected peritumorally immediately after irradiation.

All photodynamic and photodynamic glycated chitosan-treated rats hadcomplete tumor regression by the day after treatment. Tumor reoccurrencewas generally detected within 2 weeks after treatment. The efficacy ofstandard photodynamic therapy was 37.5%, which was increased followingadministration of 0.5 and 1.5% glycated chitosan with values of 82.5 and75%, respectively. Glycated chitosan significantly increased survivalrates in tumor-bearing mice compared to photodynamic treatment only(p<0.05).

In the Line 1 lung tumor model, treatment groups were as presented thetable below. mTHPC was prepared in a 2:3:5 (v/v/v) mixture of ethanol,polyethyleneglycol 400, and water for a final 0.02 mg/mL concentration.A 0.1 mg/kg dose of mTHPC was intravenously administered 24 h prior toirradiation. Animals were shielded from direct light immediately afterthe photosensitizer injection until 3 days after photodynamic treatment.Mice were restrained anaesthetized in holders exposing their backsduring light treatment. A 652 nm light from a 0.25 W diode laser wasdelivered through an 8 mm diameter liquid light guide. The power densitywas set at 110 mW/cm², fora total light dose of 30 J/cm². Immediatelyafter light irradiation, if applicable, animals were administered aperitumoral dose of 1.67% glycated chitosan. Animals were observed fortumor emergence every 2 days up to 90 days after photodynamic treatmentand 3 times a week changes in tumor size was determined.

Survival Rates After mTHPC-Based Photodynamic and Glycated ChitosanTreatment in Mice Bearing Line 1 Lung Tumors Number of Number Long-TermLong-term Treatment of Mice Surviving Mice Survival Rate (%) Control 8 00.0 Laser Treatment Only 8 0 0.0 Laser + GC 8 0 0.0 Laser + mTHPC 8 00.0 Laser + mTHPC + 8 3 37.5 1.67% GC GC = Glycated chitosan. mTHPC =meso-substituted tetra (meta-hydroxy-phenyl) chlorin-based photodynamictherapy. 0.1 mg/kg mTHPC was intravenously administered 24 h prior toirradition. 0.09 mL of 1.67% glycated chitosan was injectedperitumorally immediately after irradiation.

Tumor reoccurrences were observed in all mice within 3 weeks. FollowingmTHPC-based photodynamic therapy, administration of 1.67% glycatedchitosan resulted in a 37.5% survival rate, while other combinationswere not effective. The Line 1 lung tumor model was considered a poorlyimmunogenic tumor model. The effect of tumor-localized glycated chitosantreatment on the response of the mouse Line 1 tumors to mTHPC-basedphotodynamic therapy is presented in FIG. 6 .

The results of these studies indicate that an active immunologicalstimulation is needed to augment the efficiency of phototherapy.

Example 11

Effect of Different Components of Laser-Assisted Immunotherapy inTreatment of Metastatic Tumors in Rats

Various combinations of three components of the laser-assistedimmunotherapy system were evaluated in this study utilizing female andmale rats bearing metastatic breast and prostate tumors, respectively.The laser-assisted immunotherapy system consisted of a near-infraredlaser diode laser with a maximum output of 25 W; the laser-absorbingdye, indocyanine green; and the immunoadjuvant, glycated chitosan. Whenthe primary tumor was 0.2 to 0.5 cm³, treatment was initiated in thetumor-bearing rats. A solution of 0.2 mL of GC and/or ICG was injectedinto the center of the primary tumor in all groups. In rats receivinglaser treatment, the injections occurred 2 h before irradiation, withanimals anesthetized and the hair overlaying the tumor clipped. Thelaser settings were 2 W and 10 min, with the laser fiber tip maintaineda distance of 4 mm from the overlying skin and the laser energy directedto the treatment sites through optical fibers. The animals wereindividually housed following treatment. In the survival studies, thebreast or prostate tumor-bearing rats were observed daily and the threedimensions of each tumor were measured weekly. Female Wistar Furth rats(5 to 6 weeks of age, 100 to 125 g) were subcutaneously inoculated withthe DMBA-4 transplantable, metastatic mammary tumor (10⁵ viable tumorcells) into one inguinal fat pad of each rat. The primary tumor emerged7 to 10 days after inoculation. Metastatic tumors along the lymphaticsand at remote sites usually became palpable in approximately 2 weeks.Without treatment, tumor-bearing rats have an average survival time of35 days. Eight groups of metastatic breast tumor-bearing rats weretreated with the different components of the laser-assistedimmunotherapy system, as detailed in the table below. The survival rateand primary and metastatic tumor profiles were determined for theindividual components and various combinations of the components. Inaddition, three groups of female rats (n=16/group) were treated with0.5, 1.0, and 2.0% glycated chitosan to evaluate the impact of theimmunoadjuvant concentration on rat survival.

Treatment Parameters of Different Laser-Assisted ImmunotherapyComponents in Female Metastatic Breast Tumor-Bearing Rats Number GroupLaser Dye/Adjuvant of Rats Control — —  35^(a) ICG Injection Only —0.25% ICG^(b) 12 GC Injection Only — 1.0% GC^(b) 12 Laser Only 2W, 10min. — 12 Laser + ICG 2W, 10 min. 0.25% ICG^(b) 12 Laser + GC 2W, 10min. 1.0% GC^(b) 12 ICG + GC — 0.25% ICG/1.0% GC^(b) 12 Laser + ICG + GC2W, 10 min. 0.25% ICG/1.0% GC^(b)  31^(a) ^(a) = Data collected from 2separate experiments ^(b) = The injection volume (.0.2 mL) was injecteddirectly to the center of the primary tumor GC = Glycated chitosan ICG =Indocyanine green — = Not applicable

In the metastatic breast tumor-bearing rats, single component treatmentresulted in all rats in the indocyanine green and laser-only groupsdying, with average survival times similar to the control group. Tworats in the glycated chitosan group survived, with one rat considered along-term survivor and the other rat considered a prolonged survivor(>120 days). Following treatment with two components, 1 and 2 long-termsurvivors were observed in the laser plus glycated chitosan andindocyanine green plus glycated chitosan groups, respectively. There wasno statistical significance in the survival time when the single- ortwo-component treatment groups were compared to the control group. Ninerats had long-term survival after the three-component laser-assistedimmunotherapy (i.e., photothermal application combined with glycatedchitosan) treatment, resulting in an approximate 30% cure rate in twoseparate experiments of 31 rats. A significant difference (p<0.0001) inmedian survival time of the treated rats was observed compared to thecontrol rats. The survival rate of rats following the treatment withone, two, or three components of the laser-assisted immunotherapy systemis presented in FIGS. 7A-7C. Metastatic tumors usually emerged 2 weeksafter the inoculation of the primary tumor and reached a peak sizebefore the regression.

Example 12

Antitumor Immunity Induced by Laser-Assisted Immunotherapy and itsAdoptive Transfer

To investigate the mechanism of the antitumor immunity induced bylaser-assisted immunotherapy, adoptive transfer using immune spleencells was performed. Female Wistar Furth rats were subcutaneouslyinoculated with the DMBA-4 transplantable, metastatic mammary tumor (10⁶viable tumor cells) into one inguinal fat pad of each rat, 7 to 10 daysprior to laser-assisted immunotherapy treatment. Without treatment,tumor-bearing rats survived an average of approximately 30 days. Lasertreated rats were administered 0.2 mL of a solution containing both0.25% indocyanine green and 1% glycated chitosan directly into theprimary tumor before laser treatment. An 805 nm laser at 2 W for 10 minwas used for irradiation. The protective ability of induced immunity wasevaluated in several groups of successfully treated tumor-bearing ratsthat were challenged repeatedly with increased inoculation doses ofviable tumor cells. In addition, resistance to tumor challenges afterlaser-assisted immunotherapy and the inhibition of tumor growth wereevaluated in naive rats.

Fifteen rats that had been successfully treated by laser-assistedimmunotherapy were rechallenged with 10⁶ viable tumor cells 120 daysafter initial inoculation. Eighteen naive age-matched rats (25 weeks ofage) were inoculated with 10⁶ viable tumor cells for comparativepurposes. All of the successfully treated rats showed total resistanceto the challenge, with neither primary tumors nor metastasis observed;however, the age-matched control rats developed primary and metastatictumors and died within 30 days after inoculation. A separate group ofyoung rats (approximately 8 weeks of age) were inoculated with 10⁵viable tumor cells. Survival appeared to be dependent on the tumor dose,with control rats inoculated with 10⁶ and 10⁶ viable tumor cellssurviving on average 33 and 28 days, respectively. Successfully treatedrats usually experienced a gradual regression in both treated primarytumor and untreated metastasis.

After the first rechallenge, the rats from several experimental groupswere followed by two subsequent challenges in a time interval from 1 to5 months, with the 10⁶ viable tumor cells. The rats successfully treatedby laser-assisted immunotherapy were totally refractory to three tumorchallenges. This data is presented in the table below. In contrast, theage-matched control tumor-bearing rats developed multiple metastases inremote inguinal and axillary areas and died within 35 days. Multiplemetastases developed in all 20 control rate inoculated with 10⁵ viabletumor cells; however, these rats had a slightly increased survival timecompared with the age-matched control rats that were inoculated with thehigher 10V viable tumor cell dose. The resistance to tumor rechallengein successfully treated rats strongly suggests tumor-selective immunity.

Treatment Parameters of Different Laser-Assisted ImmunotherapyComponents in Female Metastatic Breast Tumor-Bearing Rats Number ofNumber of Tumor Death Rate Death Rate Survival Group Rats Tumor CellsRate 30 Days 40 Days (Days) Cured 15 10⁶  0%  0%  0% >120 Rats^(a) Cured15 10⁶  0%  0%  0% >120 Rats^(b) Cured Rats^(c) 15 10⁶  0%  0%  0% >120Age- 18 10⁶ 100% 83% 100% 28.2 +/− 2.8 Matched Tumor Control Rats^(d)Young 20 10⁶ 100% 20% 100% 32.7 +/− 3.5 Tumor Control Rats^(e) ^(a)=First challenge. Tumor-bearing rats cured by laser-assistedimmunotherapy, rechallenged with viable tumor cells 120 days after theinitial inoculation ^(b)= Second challenge. Tumor-bearing rats cured bylaser-assisted immunotherapy, rechallenged with viable tumor cells asecond time after the first challenge. ^(c)= Third challenge.Tumor-bearing rats cured by laser-assisted immunotherapy, rechallengedwith viable tumor cells a third time after the second challenge. ^(d)=Untreated rats the same age as the cured rats at time of inoculation (noprevious tumor exposure) ^(e)= Untreated rats that were 8 weeks of ageat the time of inoculation (no previous tumor exposure)

For the adoptive immunization experiment, viable tumor tissue harvestedfrom live DMBA-4 tumor-bearing rats was dispersed to a single-cellsuspension by grinding in a loose-fitting ground glass homogenizer. Thelong-surviving rats were sacrificed 28 days after tumor rechallenge withthe 10⁶ viable tumor cells, and their spleens were dissected free offat. Two separate experiments were conducted using the splenocytes fromcontrol tumor-bearing rats. The spleen cells were harvested 22 and 39days after tumor inoculation in the first and second experiment,respectively. Cell suspensions were prepared by mechanical disruptioninto medium with 10% fetal calf serum. Spleen cells were also collectedfrom a naive rat without prior exposure to the tumor cells. Spleen cellsand viable tumor cells were counted on a hemocytometer before mixing toa 400:1 spleen:tumor cell ratio. Naive rats were inoculated with themixture containing 4×10⁷ spleen cells and 10⁵ viable tumor cells in avolume of 0.2 mL. For the adoptive immunity transfer experiments, 4groups of naive female Wistar Furth rats were inoculated with tumorcells. The treatment groups were Group A tumor-bearing control ratsinoculated with 10⁵ viable tumor cells without treatment; Group B ratsinoculated with the tumor cells mixed with the spleen cells from acontrol tumor-bearing rat; Group C rats inoculated with the tumor cellsmixed with the spleen cells from a tumor-bearing rat successfullytreated by laser-assisted immunotherapy, 28 days after tumorrechallenge; and Group D rats inoculated with the tumor cells mixed withthe spleen cells from a naive rat without prior tumor exposure. Theexperiment was performed in duplicate and the survival of rats from bothexperiments was combined and is presented in FIG. 8 . There were noprimary or metastatic tumors observed in Group C rats indicating thatthe spleen cells from laser-assisted immunotherapy successfully treatedrats by providing 100% protection to the recipients. Multiple metastasesand death within 35 days of tumor inoculation were observed in all GroupA tumor-bearing control rats. There was no protection provided by thespleen cells from a healthy rat in Group D. A single rat out of 10 ratsin Group B survived; however, this rat later developed both primarytumor and metastases. All Group C rats were rechallenged 60 days afterthe adoptive immunity transfer, and all withstood the challenge. Theimmune spleen cells of the rats in Group C were collected and mixed withtumor cells in the same ratio as in the first adoptive transfer toevaluate the ability of these animals' spleen cells in protecting asubsequent cohort of normal Wistar Furth recipient rats (n=6) that wereinoculated with this admixture. The immune cells from the Group C ratsprotected 5 of 6 naive rats, as neither primary nor metastatic tumorswere observed. The rat that died had a prolonged survival time (60versus 30 days) and a delayed tumor emergence after inoculation (37versus 7 to 10 days), in comparison with the control group.

The resistance of successfully treated rats when tumor rechallengedstrongly suggests that the tumor-selective immunity has a long-lastingeffect.

Example 13

Combination of Laser-Assisted Immunotherapy and Low-Dose Chemotherapy

In one exemplary clinical study, two breast cancer patients receivedcyclophosphamide weekly (after inCVAX treatment) at a dose of between150 and 200 mg/m². The patients initially responded well to thetreatment with tumor shrinkage and minimal adverse reactions. After afew months the response slowed, so the oncologist changed the low-dosechemotherapy to a weekly regimen of Paclitaxel at 75 mg/m², and againthe response was very good with shrinking tumors. No new metastasesappeared. The patients continued to receive the low dose chemotherapy. Athird patient became operable following the low dose chemotherapy, and amastectomy was performed, so a combination with surgery was also anoption.

Example 14

Demonstration of the Sterile Filterability of IP-001

IP-001 is an illustrative embodiment of Glycated Chitosan (GC), which isa semi-synthetic glucosamine-based polymer. IP-001 is a novel andunobvious GC. Specifically, the data below supports the advantageous andunexpected properties of IP-001 with respect to its ability to bemanufactured in a consistent and compliant manner. The IP-001 isformulated as a 1.0% solution (w/w) in water buffered to pH of 5.5 andhas a viscosity of 50-60 cPs and is meant for intratumoral injection.IP-001 is a variant of GC and has the following molecularcharacteristics:

-   -   Weight Averaged Molecular Weight (Mw) of ˜250 kDa    -   Degree of Deacetylation (DDA) of ˜80%    -   Degree of Glycation (DoC) of ˜5%

One of the main advantages exhibited by IP-001 is its ability to besterile filtered. The sterile filtration of pharmaceutical solutions isan industry standard for ensuring patient safety. Specifically, in thearea of sterile injectable solutions, sterile filtration is often thefavored method for sterilization, as it is an easily scalable processand does not affect the chemical structure of the active pharmaceuticalingredient (API) as often occurs during autoclave- or gammairradiation-based sterilizations. Additionally, sterile filtrationoffers cost advantages in the development, validation and execution ofthe process, relative to autoclave and gamma sterilization. The sterilefiltration of solutions of polymers adds an additional degree ofcomplexity, as physiochemical properties, such as viscosity can oftenslow or stop the filtration process. Therefore, the conditions of thefiltration as well as the chemical and physiochemical characteristics ofthe polymer must be considered carefully.

With respect to IP-001, it was unexpectedly discovered that the specificembodiment in conjunction with a formulation including defined ranges ofconcentration and pH were needed to successfully sterile filter theformulation and provide a compliant and consistent drug product. Asshown below, the results of our experiments demonstrate thefilterability compared to embodiments of GC that are outside of thepreferred ranges, described above and specifically include those with amolecular weight of less than 500,000 Daltons.

It should be noted that the state of the art, an example of which beingMirko Weinhold's published PhD thesis entitled: “Characterization ofChitosan using Triple Detection size-exclusion chromatography and ¹³CNMR spectroscopy” at the Center for Environmental Research andSustainable Technology, University Bremen, October 2010), teaches thatchitosan viscosity increases in a linear fashion as the molecular weightof the GC increases. Indeed, we discovered unexpectedly after many yearsof trial and error that the prior art teachings, if followed, do notprovide sufficient guidance which would lead one to conclude thatparticle size correlates to molecular weight (see specifically the datapresented below).

Under current regulatory and scientific standards, pharmaceuticalsolutions can be considered sterile following the filtration through afilter with an effective pore-size of 0.22 microns or smaller.Additionally, the process and materials must be tested and validated ina GMP-compliant manner. The sterile filtration of IP-001 drug producthas been carefully studied. The full-scale process for the sterilizationof IP-001 utilizes Pall Corporations Flurodyne capsule filters (part#KA2DFLP1S) in a redundant (serial) manner. The filter chosen meets allregulatory requirements and is chemically compatible with IP-001.Additionally, a product-specific validation of the process (Study#-VAL-AM-000754-B) was carried out. As part of this study, IP-001 drugproduct demonstrated multiple times its ability to effectively undergosterile filtration.

Referring to FIG. 9 (recirculation data for IP-001 Drug Product), thedata clearly shows that when IP-001 drug product is recirculated throughsterilizing-grade membranes for up to 3 hours at a constant pressurethere is minimal loss in flow rate (indicating minimal fowling orclogging of the filter). This test represents an extreme stressing ofthe system, as sterile filtration in practice is only a single ourdouble pass and not a continuous recirculation of the solution throughthe membrane. This data strongly supports the fact that IP-001 DrugProduct can be filter sterilized with little to no loss in integrity ofthe polymer solution.

The process validated by Pall Corporation in Study VAL-AM-000754-B wassubsequently performed on scale multiple time. In one example, theproduction of GMP-grade IP-001 Drug Product, the following data wascollected:

-   -   Pre-filtration weight of IP-001 Drug Product—7.602 kg    -   Time for redundant sterile filtration—3 hours    -   Post-filtration weight of IP-001 Drug Product—7.384 kg    -   Yield of filtration—97.1%

in order to demonstrate of the advantage of IP-001 over other lesspreferred GC embodiments with respect to sterile filtration, a directcomparison of sterile filtration of IP-001 and larger Mw (500 kDa) GCwas performed.

Comparison with Song et al. (Immunopharmacology and Immunotoxicology,2009; 31 (2), 202-208)

A 1% solution of GC with a Mw of approximately 500 kDa was synthesizedas described in Song, et. al. Song describes a sterilization byautoclave, and it was the opinion of Immunophotonics that this processwould in fact affect the MW of the polymer. To test this, solutions of500 kDa GC were tested for their abilities to be sterile filtered bothbefore and after the reported autoclave sterilization and compared tothat of IP-001 Drug Product.

Referring now to FIG. 10 , which shows filtration rate data for various1% solutions of GC. In order to generate the data in FIG. 10 , 1 mL ofeach solution was added to a 2.5 mL syringe fitted with a luer-fitteddigital pressure sensor. A small scale, representative sterile filterwith a luer fitting was then attached to the outlet of the pressuresensor. The solutions were forced through the filters keeping thepressure between 500 and 600 psi. The resulting drop-rate was measured.

The data in FIG. 10 clearly shows that the IP-001 drug product (lotIPDP-2015001) maintained a consistent flow rate until all the solutionhad been pushed through the filter. In contrast, the pre- andpost-autoclave solutions of 500 kDa GC exhibited steadily decreasingdrop rates, both ultimately clogging the filters and thus halting thefiltration. Additionally, the data corresponds with the hypothesis thatautoclaving reduces the molecular weight, as shown by the lowerpressures and improved flows for autoclaved materials when compared tonon-autoclaved material.

Referring now to FIG. 11 , particle size data was collected for the 3samples tested. A convenient estimate of particles sizes for chitosansolutions is the radius of gyration (Rg). While Rg is not the exactradius of the particle, more often than not, it is only slightly lessthan the radius of the particle. The data collected for the Rg of the GCsolutions provides an explanation for their observed behavior in thefiltration experiments. The radius of gyration for IP-001 Drug Productwas measured to be ˜32 nm while both solutions of the Song 500 kDa GCexhibited Rg's of ˜52 nm. When the larger end of the polymer range isconsidered, it becomes evident why the material taught by Song et al.would not sterile filter, as the particles are approaching or largerthan the effective pore size of the sterilizing filter.

In conclusion, the data described herein clearly demonstrates theadvantage of the new and unobvious IP-001 with respect to its sterilefilterability when compared to the GC reported by Song et al. and byextension, GC's of larger molecular weights. Additionally, andunexpectedly, IP-001 represents an optimal form of GC for sterilefiltration. It is known that lowering the pH of solutions of chitosanincreases the Rg while increasing the pH of GC solutions causes thematerial to crash out of solution (i.e. precipitate). Therefore, it wasunexpectedly discovered altering the pH of larger GC molecules would notallow for sterile filtration. The data described herein and theadditional development work performed for IP-001 clearly support thatthe described embodiment represents and clear and unexpected advantagewhen compared to the prior art.

Other Embodiments

From the foregoing description, it will be apparent to one of ordinaryskill in the art that variations and modifications may be made to theembodiments described herein to adapt it to various usages andconditions.

We claim:
 1. A method of treating a cancer in a patient in need thereof,comprising administering by injection a pharmaceutical formulationcomprising a glycated chitosan to the patient, wherein: the glycatedchitosan has a molecular weight of about 100,000 to about 300,000Daltons; the glycated chitosan has a degree of deacetylation of about75% to about 99%; and the glycated chitosan has a degree of glycation offree amino groups of about 0.1% to about 30%.
 2. The method of claim 1,wherein the cancer comprises a neoplasm.
 3. The method of claim 2,wherein the pharmaceutical formulation is administered into theneoplasm.
 4. The method of claim 3, wherein the method comprisesablating the neoplasm.
 5. The method of claim 1, wherein the cancer is asolid tumor cancer.
 6. The method of claim 2, wherein the cancer ismetastatic.
 7. The method of claim 1, wherein the glycated chitosan hasa molecular weight of about 250,000 Daltons.
 8. The method of claim 1,wherein the glycated chitosan has a degree of deacetylation of about80%.
 9. The method of claim 1, wherein the degree of glycation is about2%, about 7%, or about 12.5%.
 10. The method of claim 1, wherein thepharmaceutical formulation comprises about 0.5%, about 1%, or about 1.5%by weight of the glycated chitosan.
 11. The method of claim 10, whereinthe pharmaceutical formulation comprises about 1% by weight of theglycated chitosan.
 12. The method of claim 1, wherein the pharmaceuticalformulation is sterile filtered.
 13. A method of generating an in situautologous vaccine, comprising: ablating a neoplasm in a host, therebyreleasing fragmented neoplastic tissue and cellular molecules; andintroducing by injection a pharmaceutical formulation comprising aglycated chitosan into the neoplasm, wherein: the glycated chitosan hasa molecular weight of about 250,000 Daltons; the glycated chitosan has adegree of deacetylation of about 80%; and the glycated chitosan has adegree of glycation of free amino groups of about 2%, about 7%, or about12.5%.
 14. The method of claim 13, wherein the neoplasm is a cancer is asolid tumor cancer.
 15. The method of claim 14, wherein the cancer ismetastatic.
 16. The method of claim 13, wherein the pharmaceuticalformulation comprises about 0.5%, about 1%, or about 1.5% by weight ofthe glycated chitosan.
 17. A method of enhancing immune system responseto an antigen in a patient in need thereof, comprising administering apharmaceutical formulation comprising a glycated chitosan to a patient,wherein: the glycated chitosan has a molecular weight of about 250,000Daltons; the glycated chitosan has a degree of deacetylation of about80%; and the glycated chitosan has a degree of glycation of free aminogroups of about 2%, about 7%, or about 12.5%.
 18. The method of claim17, wherein the antigen is a tumor antigen.
 19. The method of claim 18,wherein the tumor is a solid tumor cancer.
 20. The method of claim 17,wherein the antigen is an infectious agent antigen.
 21. The method ofclaim 17, wherein the pharmaceutical formulation comprises about 0.5%,about 1%, or about 1.5% by weight of the glycated chitosan.